Methods for detection of botulinum neurotoxin

ABSTRACT

Provided herein is a large immuno-sorbent surface area assay (ALISSA) for the rapid and sensitive detection of botulinum neurotoxins (BoNTs) and anthrax toxin. This assay is designed to capture a low number of toxin molecules and to measure their intrinsic protease activity via conversion of a fluorogenic or luminescent substrate. Also provided herein are novel peptides that can be specifically cleaved by BoNT and novel peptides that are resistant to cleavage by BoNT. The combination of these cleavable and control peptides can be used for implementation of an exemplary ALISSA used to specifically detect BoNT enzymatic activity. Furthermore, the ALISSA as described herein may also be used in a column based format for use in a high-throughput system for testing large quantities of samples.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.13/600,186 filed Aug. 30, 2012, which is a continuation-in-part of U.S.patent application Ser. No. 13/306,769, filed Nov. 29, 2011; which is adivisional U.S. patent application Ser. No. 12/134,092, filed Jun. 5,2008, now issued as U.S. Pat. No. 8,067,192; which claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/942,199, filed Jun. 5,2007, all of which are incorporated herein by reference.

GOVERNMENT INTEREST

The present invention was supported by National Institutes of Healthgrant AI-65359. The government may have certain rights in the presentinvention.

BACKGROUND

Botulinum neurotoxins (BoNTs) are important medical and cosmetic agents,used to treat dystonias, blepharospasms, hyperhidrosis, and otherneurological diseases. However, BoNTs also represent the most toxicsubstances known and their potential abuse as a threat agent is feared(Arnon 2001; Wein 2005). The detection of Botulinum neurotoxin (BoNT) incomplex samples such as foods or clinical specimens represents ananalytical challenge. The current “gold standard” in the art fordetecting BoNT is the mouse toxicity assay, which can detect as littleas 10 pg BoNT (Ferreira 2003). However, BoNT can be lethal to humans insystemic doses as low as 1 to 2 ng/Kg body weight (Arnon 2001).Therefore, there is a need in the art for more sensitive assays fordetecting the presence of BoNT in a sample.

BoNTs have gained popularity as cosmetic drugs, and have also beensuccessfully used for the treatment of a variety of neurological andneuromuscular disorders (Schantz 1992; Johnson 1999). Productscontaining BoNT are approved for the medical treatment of severaldiseases, i.e. cervical dystonia, torticolis, blepharospasm,hyperhydrosis, strabismus and migraines. Further, the products BOTOXCOSMETICS®, VISTABEL®, BOCOUTURE® and AZZALURE® are licensed as ananti-wrinkle treatment. With the ever-increasing medical use of BoNT,its sensitive and specific detection in manufacturing processes as wellas clinical research laboratories is of crucial importance. Accordingly,the potency of each batch of BoNT must be determined by the manufacturerbefore release to ensure the safety and efficacy of the product. Thistesting is widely performed using the classical mouse LD50 assay, whichmeasures the potency of each batch of the product by determining thedose that will kill 50 percent of the animals. Animal testing for eachbatch of BoNT is expensive, slow, provides limited throughput, andrequires sacrificing animals. Also, because of the lack of astandardized testing procedure, the units of biological activity areoften unable to be directly converted into precise doses for human use,and overtreatment with BoNTs can cause iatrogenic forms of botulism(Partikian 2007; Crowner 2007). Thus, there is a need for an alternativemethod for testing products containing BoNT that is more efficient andcost effective.

Natural BoNT resides within ˜300, 500 or 900-kDa protein complexestogether with other non-toxic components, the neurotoxin associatedproteins (NAPs) (Sakaguchi 1982; Chen 1998; Sharma 2003; Melling 1988;Zhang 2003; Aoki 2001). Several structurally distinct serotypes of BoNT(types A to G) have been discovered. BoNT Type A (BoNT/A) is mostprevalent in the Western United States (Smith 1978) and is causativelyinvolved in approximately 60% of the IB cases in California (the restbeing mostly attributed to type B) (Arnon 2001). The toxin itself is a150-kDa zinc-binding metalloprotease that, following expression, isendogenously cleaved into a 100-kDa heavy and a 50-kDa light chainconnected by a reducible disulphide bond (Schiavo 2000) and by abelt-like extension of the heavy chain that loops around the light chain(Lacy 1998). The catalytic site is located on the light chain (Kurazono1992). Reduction of the chain-bridging disulphide bond exposes thecatalytic site and enhances its activity (Lacy 1998), also referred toas “activation” of the toxin by some authors and toxin manufacturers(Cai 1999; Cai 2001). The potency of BoNT results from its ability tocleave on or more of the three SNARE proteins involved in fusingacetylcholine-containing synaptic vesicles with terminal motor neuronsmembrane, triggering muscle contraction (Shiavo 2000).

Detection of low levels of BoNT in a sample using prior art methods isdifficult. However, due to the enormous potency of the toxin, which canbe lethal for humans in systemic doses of 1 to 2 ng/Kg body weight(Arnon 2001), these low levels can be extremely dangerous. For example,in infant botulism (IB), a condition in which a baby's intestines havebecome colonized by toxin-secreting Clostridium botulinum bacteria, itis possible to detect BoNT in stool samples (Arnon 2006). However,attempts to diagnose IB serologically via detection of BoNT in the bloodhave been deemed unreliable (Schantz 1992). Nevertheless, the systemicpresence of the toxin in IB cannot be disputed, because of its apparentquick distribution throughout the infant's entire body, by which itefficiently shuts down motor neurons distant from the intestinal source.The resulting symptoms can include complete paralysis and respiratoryfailure.

The definite diagnosis of botulism requires detection of BoNTs inclinical specimens. Most commonly used and relied on is the life mouseassay. This assay can detect as little as 10 pg BoNT (Ferreira 2003). Inthe life mouse assay, mice are injected intraperitoneally (i.p.) with0.5 mL/mouse of sample, treated with type A or B antitoxin, and observedfor signs of botulism or death, typically over a 48 hour period.Toxicity is expressed by the number of hours until death (Kautter 1977;Sharma 2006). As in many animal experiments, the results of the mouseassay may vary. Four- to five-fold differences in response to a givendose are typical (Sugiyama 1980). Other and generally faster methods forBoNT detection include use of fluorescence resonance energy transfer(FRET) substrates for BoNT (U.S. Pat. No. 6,504,006), variousenzyme-linked immunosorbent assays (ELISAs) (Sharma 2006),Enzyme-amplified protein micro arrays with a “fluidic renewable surfacefluorescence immunoassay” (Varnum 2006), mass spectrometric assays (Barr2005; Kalb 2005; Boyer 2005; Kalb 2006), immuno-PCR detection (Chao2004), and recently, a real-time PCR-based assay that utilizes reporterDNA-filled liposomes which bind to immobilized BoNT/A via gangliosides(Mason 2006a; Mason 2006b). Reported detection limits and sample typesfor these various methods are summarized in Table 1. Except for thePCR-based assays, most assays are not well suited to provide the desireddetection of less than 1 pg/mL BoNT in a complex sample. Byapproximation, 1 pg/mL corresponds to the lethal concentration underpresumed equal distribution throughout the human body.

TABLE 1 Reported performance of existing Botulinum toxin assaysDemonstrated for Sensitivity Test method Sample Type (fg/mL) Assay TimeMass spectrometry milk, serum, stool 320,000 <4 hrs (Endopep-MS)²²⁻²⁵extract Enzyme-linked liquid and solid 60,000 6-8 hrs immunosorbentassays foods, serum (ELISA)¹⁹ ELISA-HRP²⁹ therapeutic 9,000 4-6 hrspreparations Mouse assay (gold foods, serum, ~6,000 typically 48 hrsstandard)¹⁸ stool Enzyme-amplified blood, plasma 1,400 <10 min. perprotein microarray and measurement fluidic renewable surfacefluorescence immunoassay²¹ Immuno-PCR²⁶ carbonate buffer 50 4-6 hrsImmuno-PCR with deionized water 0.02 6 hrs ganglioside-mediated liposomecapture^(27,28)

Anthrax lethal factor (LF) is another zinc metalloprotease that has beensuccessfully been adapted for use in the ALISSA. LF constitutes one ofthe three components of anthrax toxin that is produced by Bacillusanthracis, together with protective antigen (PA) and edema factor (EF)(Brossier 2001). LF specifically cleaves members of themitogen-activated protein kinase kinase (MAPKK) family, leading to theinhibition of essential signaling pathways. LF alone is not toxic; itrequires the presence of PA for its translocation into cells (Brossier2001). Macrophages are believed to be primarily affected by LF (Hanna1993). A specific and sensitive assay for the detection of LF ispotentially useful for early diagnosis of anthrax infection and isexpected to be a useful research tool to advance the understanding ofthe mechanism of action of anthrax toxin (Boyer 2007).

SUMMARY

Methods are provided for detection of BoNT in complex biological sampleswith high sensitivity and specificity. In certain of these embodiments,the methods are based on specific affinity enrichment of a target toxinor target enzyme (“target”) onto a solid support followed byfluorometric or luminescent readout. In certain of these embodiments,the assays have a sensitivity of at least about 0.5 femtograms targetper one mL sample or about 300 target molecules per sample. In certainof these embodiments, the solid support is a bead matrix that containsimmobilized, anti-enzyme-specific antibodies and/or anti-toxin-specificantibodies. In other embodiments, binding (also referred to as“capturing”) and/or immobilization of the target toxin or enzyme is suchthat the toxin's or enzyme's activity on its substrate is accelerated.Products and methods may increase enzymatic activity and/or turnoverrate when the enzyme is preconjugated to the bead. Such modificationsmay further allow for enhanced sensitivity and speed of the assay.

In certain embodiments, binding and/or immobilization of the targettoxin or enzyme is achieved without resulting in inactivation orreduction of the target activity on its substrate. For example,antibodies may be BoNT-specific antibodies that bind BoNT but do notinactivate BoNT-specific enzymatic activity. In certain embodiments,antibodies may specifically target the catalytic BoNT light chains andbind the BoNT light chain without significantly inhibiting BoNTenzymatic activity. Also, antibodies may be BoNT-specific antibodiesthat bind BoNT such that the BoNT activity is accelerated.

In certain of these embodiments, the fluorometric readout is based onspecific cleavage of a fluorogenic substrate. For example, BoNT-specificcleavage of a fluorogenic BoNT substrate such as the SNAPtide describedin U.S. Pat. No. 6,504,006 as well as other coumarin derivatives isuseful in certain embodiments. Additional suitable substrates includevarious soluble NSF attachment protein receptor (SNARE), or one or morefluorogenic toxin or enzyme peptide substrate.

The methods provided herein may be used to detect BoNT type A, B, C, D,E, F, and/or G, or their subtypes. In certain embodiments, methods areprovided for detection of BoNT serotypes including subtypes withattomolar sensitivity. The assays include use of a BoNT serotype A assaywith a large immuno-sorbent surface area (BoNT/A ALISSA) that hasattomolar sensitivity in biological samples such as complex samples,serum and liquid foods.

In other embodiments, luminescent based readout assays are provided fordetection of BoNT. The methods include use of bioluminescent BoNT/A andBoNT/B substrates including novel engineered variants of recombinantluciferase proteins. In some embodiments, the bioluminescent substratesinclude serine-glycine and glycine linkers to optimize the turnover ratefor BoNT cleavage of the substrates. In certain embodiments,bioluminescent assays include use of luminescent proteins able to emitlight at multiple wavelengths for multiplexed simultaneous detection ofone or more serotype.

In certain embodiments, the methods and assays include development anduse of novel fluorogenic or bioluminescent substrates for toxin orenzyme detection. Such novel substrates include those having resistanceto non-BoNT proteases while remaining cleavable by the target toxin orenzyme. The novel substrates may include fluorogenic synthetic peptidesthat can be cleaved by the BoNT/A or BoNT/B light chain independent ofthe BoNT heavy chain. In certain of these embodiments, these fluorogenicsubstrates can also be used as a toxin-free positive control for theimplementation of an assay used to detect BoNT enzymatic activity.

The methods and assays provided are broadly useful and as such may beused to detect a wide variety of toxins and/or other enzymes such asanthrax lethal factor, human chitinases (e.g. CHIT1 or AMCase) andproteases such as fungal protease Pep1 and Pep2 from Aspergillusfumigatus, and other toxins exhibiting zinc metalloprotease activity,among other enzyme targets. The assay with a large immuno-sorbentsurface area (ALISSA) methods may be expanded for use in detection ofany toxin or enzyme including all BoNT serotypes.

The systems, methods and kits provided herein may be used to detectand/or measure toxin or enzyme levels in a variety of samples. Incertain embodiments, the methods may be used to measure toxin or enzymedistribution in systemic circulation, in a biological fluid sample,cell, tissue and/or organ of an animal or human. The sensitivity,specificity, speed and simplicity of the methods provided herein areparticularly useful for diagnostic, biodefense and pharmacologicalapplications.

In other embodiments, the methods and kits provided herein may be usedto evaluate the potency or efficacy of products containing BoNT. Forexample, the methods and kits may be used to evaluate products thatcontain, but are not limited to, active ingredients such asonabotulinumtoxinA, rimabotulinumtoxinB, abobotulinumtoxinA andincobotulinumtoxinA to measure the functional activity of the toxinprotein. In some embodiments, the method herein can be used to confirmthe amount of active ingredient contained in the product, which isuseful is a pharmacological production setting, such as a lab or aplant.

The systems and methods provided herein may also be used to detectinhibitors that prevent BoNT cleavage activity. In one embodiment, themethods provided herein can be used as a high-throughput detectionsystem for inhibitors of BoNT enzymatic activity. Such high-throughputdetection system is preferably automated for large-scale detection andtesting, such as may be used in a diagnostic medical laboratory or in amanufacturing facility.

In addition to the exemplary embodiments described above, furtherembodiments and aspects will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1(A) shows synthesis of the immuno-affinity matrix for BoNTenrichment. Protein A sepharose beads are coupled to affinity purifiedanti-BoNT antibodies. The FC domain of the antibodies is cross-linked tothe protein A using disuccinimidyl suberate (DSS). Non-cross-linkedantibodies are removed through stringent washing. FIG. 1(B): Cleavage offluorogenic substrate by immuno-affinity enrichment of BoNT/A.

FIG. 2(A) shows immobilized polyclonal rabbit antibody does notsignificantly inhibit specific proteolytic activity of BoNT/A and FIG.2(B) shows western blot analysis of BoNT/A using anti-Clostridiumbotulinum A toxoid antibodies. The antibody recognized both heavy (H)and light (L) chain of the toxin.

FIG. 3A-E shows an optimization of assay parameters. (A) BoNT/Aconcentration-dependent cleavage of SNAPtide after one hour reactiontime in 1 ml 10% FBS. (B) SNAPtide (25 μg/ml) conversion time-curve byBoNT/A. (C) Effect of the number of beads exposed for 3 hours to 1 ml10% FBS spiked with BoNT/A. (D) Time course of BoNT/A enrichment on thebeads. (E) Effect of temperature on binding of bead-immobilized BoNT/A(from complex) at one hour incubation time.

FIG. 4A-B shows a determination of assay performance. (A) Detection ofBoNT/A from two different commercial sources, Metabiologics and the ListBioLabs in serum spiked with serially diluted toxin. “Pre-act” indicatestoxin pre-activation in 5 mM DTT. (B) Detection of BoNT/A inrepresentative complex samples. The samples were spiked with undilutedhuman serum, carrot juice, reconstituted non-fat powdered milk, freshmilk and GP-diluent.

FIG. 5A-B shows an evaluation of monoclonal mouse antibodies for ALISSA.FIG. 5A shows an ALISSA with BoNT/A complex-spiked pooled human serumusing antibodies to HC (F1-5) and LC (F1-40), protein A agarose beads(Pierce) and #115 peptide (City of Hope). FIG. 5B shows a bead-freereaction of the substrate in presence or absence of the USDA antibodies;100 pM BoNT/A complex was used where indicated.

FIG. 6A-B shows an evaluation of horse antibodies for ALISSA. FIG. 6Ashows a BoNT/A ALISSA in pooled human serum using equine polyclonal antiBoNT/A antibody (CDC, lot #00-0056L, provided by Dr. Steve Arnon at theCDPH), protein NG agarose beads (Santa Cruz) and SNAPtide (ListBiological Laboratories). FIG. 6B shows a bead-free reaction with equalamount of equine antibody and 100 pM BoNT/A complex (Metabiologics).

FIG. 7 shows the results of an ALISSA performed using 5 μg ofanti-BoNT/A antibody with decreasing concentrations of BoNT/A complex(10⁻⁹ to 10⁻¹⁷ mol/L) and approximately 120,000 beads.

FIG. 8A-C shows results of BoNT/A substrate cleavage using single ordouble affinity immunomatrices. Beads bound to F1-40 (BoNT/A LC, mab)(left bar), anti-FITC (goat) (middle bar), or a combination of F1-40 andanti-FITC (right bar), were incubated with BoNT/A specific substrate andsupernatant containing BoNT/A complex. Fluorescence of the cleavedsubstrate was measured after overnight (FIG. 8A) or 48 hour (FIG. 8B)incubation. A similar experiment was performed with no beads (onlysupernatant containing BoNT/A complex and BoNT/A specific substrate),and fluorescence of the cleaved substrate was measured after 48 hours(FIG. 8C).

FIG. 9 shows a titration assay to determine concentrations of antibodyto use for an optimal signal to noise ratio for the double affinityimmobilization matrix. The titration experiment was performed using 10pM BoNT/A complex in 1 mL pooled human serum with 120,000 beads. 0.5 μgof anti-BoNT/A LC antibody was used with increasing concentrations ofanti-FITC antibody (0.0-2.5 μg). The ratio of anti-FITC to anti BoNT/ALC ranged from 0-5.

FIG. 10A-D shows a comparison of the specificity, sensitivity andkinetics of bead-based ALISSA (10A) and bead-free assays (10B). FIGS.10C and 10D illustrate the hydrolysis of SNAPtide by BoNT/A by a linearrelationship between the reciprocal substrate concentration and theactivity of the enzyme in bead-based ALISSA and bead-free assays,respectively.

FIG. 11 shows a line graph demonstrating the sensitivity of the BoNTALISSA compared to the “gold standard” mouse bioassay. The BoNT ALISSAis 4-5 orders of magnitude more sensitive than the mouse assay. Thenumbers shown in boxes represent the mouse LD₅₀ calculated per injected0.5 mL sample. One LD₅₀=30 pg BoNT/A complex.

FIG. 12 shows a schematic of one embodiment of a BoNT ALISSA. Antibodiesagainst targets, such as BoNTs or anthrax lethal factor, are conjugatedon protein A-coated beads via their Fc regions and then cross-linked. Animmobilized toxin protease molecule cleaves fluorogenic reportermolecules and releases unquenched fluorescent products.

FIG. 13 shows a standard curve of the fluorescence signal of theunquenched calibration peptide, which is structurally identical to theFITC-containing cleavage product resulting from BoNT/A hydrolysis ofSNAPtide by BoNT/A; “y” in RFU; “x” in nM; “R” is the correlationcoefficient.

FIG. 14 shows the spectrums of the fluorophores (5-FAM and 4-MU) andquencher (DABCYL) of the BoNT cleavable and control peptides. Uponexcitation, the 5-FAM fluorophore (Ex/Em=535 nm/485 nm) and the 4-MUfluorophore (Ex/Em=466 nm/350 nm) will be quenched by DABCYL if thepeptide is not cleaved.

FIG. 15 shows an analysis of alternative fluorogenic BoNT/A substratesas compared with commercial SNAPtide. About 5 μM substrate solutionswere incubated with 2 nM BoNT/A complex for 1 hr at 37° C. 1 to 5indicate COH-made synthetic BoNT/A and their sequences [1—SEQ ID NO:20(#110); 2—SEQ ID NO: 21 (#115); 3—SEQ ID NO:19 (#112); 4—SEQ ID NO:22(#116); 5—SEQ ID NO:5 (#113); 6-commercial SNAPtide]; DABCYL is4-(dimethylaminoazo)benzene-4-carboxylic acid conjugated to the ε-aminogroup of lysine.

FIG. 16 shows the remarkable specificity of novel BoNT cleavable andcontrol peptides. BoNT cleavable peptides (#115; SEQ ID NO: 21 and #116;SEQ ID NO: 22 and control #110, 111, 112, and 113; SEQ ID NOS: 20, 23,19, and 5, respectively) showed some cleavage upon addition of trypsin(left panel). However, control peptides (#110-113) were not cleaved byBoNT/A complex (middle panel); whereas, BoNT/A cleavable peptides (#115and #116) were cleaved by BoNT/A complex. Control peptide #112 was notcleaved by either serotype BoNT/A or BoNT/B (right panel).Significantly, BoNT/A cleavable peptide was only cleaved by BoNT/Aserotype and not BoNT/B serotype (right panel), which demonstrates thatBoNT/A cleavable substrate is highly specific for BoNT/A. CommercialSNAPtide (#521) was cleaved by trypsin and BoNT/A complex (left andmiddle panels).

FIG. 17 shows Michaelis-Menten kinetics of BoNT/A cleavable substrates(#115 (SEQ ID NO: 21) and #116 (SEQ ID NO: 22)) and control substrates(#112 (SEQ ID NO: 19) and #113 (SEQ ID NO: 5)) with BoNT/A, trypsin,proteinase K, and thermolysin. Enzymes (10 nM) were incubated for 2hours with different concentrations of BoNT/A cleavable and controlsubstrates. The velocity of the enzyme reaction is fit as a function ofsubstrate concentration. Peptide cleavage obeys Michaelis-Mentenkinetics.

FIG. 18 shows linear relationships between the signal responses of 4-MUand 5-FAM substrates when tested with different enzymes (proteinase K,trypsin, and thermolysin). BoNT/A enzyme concentration tested rangedfrom 1 pM to 100 pM.

FIG. 19A-D shows cleavage of 5-Fam- (A) or 4-Mu-containing (B) peptidesby LC/A subtypes. LC A4 does not cleave BoNT/A-cleavable peptides #115(A) and #116 (B). Less than 2% of the commercial SNAPtide (#521) wascleaved by LC A4. Peptide solutions (5 μM) were incubated with LCs (20nM) at 37° C. Western blot analysis of recombinant SNAP25 cleaved (c) byLC A4 compared to its un-cleaved (u) form (C). ALISSA performance of5-Fam-containing BoNT/A-cleavable (#115) and control (#112) peptides inthe presence or absence of Zn²⁺-metallo-protease inhibitor, TPEN (100μM) (D). Rabbit polyclonal to BoNT/A toxoid antibody was used to bindBoNT/A complex from spiked human serum with protein NG-agarose beads.

FIG. 20 shows a schematic and matrix-assisted laserdesorption/ionization (MALDI) mass spectrum for the novel BoNT/B ALISSAsubstrate. The BoNT/B specific substrate (SEQ ID NO: 24) contains aminoacid residues 59-78 of the VAMP protein that is specifically cleaved byBoNT/B at the expected site of natural VAMP cleavage, between theglutamine and phenylalanine. Both the fluorophore, 5-carboxyfluorexcein(5-FAM), and the quencher, 4-dimethylamino phenyl azo enzoic acid(DABCYL), were placed on the ε-amino groups of lysine residues that arepart of the natural VAMP sequence, allowing for a non-inhibited enzymesubstrate interaction. High specificity cleavage of the substrate byBoNT/B was indicated using MALDI-MS resulting in product at m/z 2245.98and m/z 1528.88. The uncleaved substrate produced a peak at m/z 3755.81.

FIG. 21A-C shows the remarkable BoNT/B substrate specificity for thenovel substrate for use in BoNT/B ALISSA (SEQ ID NO: 24). The BoNT/Bsubstrate was incubated with either 10 nM recombinant BoNT LC/A or LC/Bat 37° C. Specificity of the BoNT/B substrate to BoNT/B LC versus BoNT/ALC was measured fluorometrically (A). The reaction was also performedusing BoNT/B complex that was immobilized with anti-BoNT/B antibody in abead-based assay (B). The bead based assay has a remarkably moresensitive detection limit of BoNT/B substrate cleavage compared with thebead free reaction (C).

FIG. 22 shows BoNT/E ALISSA with the commercial substrate, SNAP Etide(List Biological Laboratories). The reaction was performed using BoNT/Ecomplex (Metabiologics), immobilized with anti-BoNT/E antibody(Metabiologics) on protein A/G beads (Santa Cruz). The bead-based ALISSAhas a limit of detection of ˜1 fmol/L BoNT/E complex, whereas the limitof detection for the bead-free reaction is >10 pmol/L.

FIG. 23 shows SDS-PAGE analysis of recombinant human SNAP-25 proteolysisreaction in presence of BoNT/A. The gel shift is indicated by the arrow.E. coli protein impurities are denoted by asterisk. The small cleavageproduct contains the C-terminus of rSNAP25 with the hexahistidine tagand measure about 4 kDa in size, resulting in light-emitting E. coliexpressing his-tagged FFL.

FIG. 24 shows cloning and expression of engineered firefly luciferase(FFL) fusion proteins for the bioluminescent detection of BoNT/A. TheSDS gel is an example that demonstrates the progress in the purificationof full length FFL[1-550], control, and an N-terminal FFL fragment[1-475] fused to the BoNT/A-cleavable portion of a SNAP25 sequence(residues 187-206).

FIG. 25 (A) an exemplary schematic of a synthesis scheme forconstructing recombinant overlapping split FFL having a SNAP25-sequenceinsert (SEQ ID NO: 1). As depicted, two PCR products are generated usinga yeast plasmid (pGAL-FFL) as a template and then cloned subsequentlyinto the same pETBlue-2 vector. The BoNT/A cleavage site for SNAP25(amino acid residues 187 to 206; SEQ ID NO: 6) is embedded into thesequence via synthetic primers (SEQ ID NO: 7 and 8) using acodon-optimized sequence for E. coli. Similar synthesis schemes areemployed for constructing split FFL fusion proteins for targeted SNAREsequence of other BoNT serotypes. FIG. 25 (B) shows restriction analysisof the intermediate plasmid depicted in (A) (plasmid “1”; SEQ ID NO: 9)and the final product (plasmid “2”; SEQ ID NO: 10). The identifier “1”is the uncut pETBlue-2 FFL[1-475] vector; “1 cut” is “1” cut with NcoIand KpnI; “2” is the uncut final productpETBlue-2FFL[1-475]-SNAP-FFL[265-550] and “2 cut” is the “2” cleavedwith NcoI and PvuII.

FIG. 26A-B shows schematic representations of two strategies forbioluminescent detection system. (A) shows an assay comprising a DualChamber system; (B) shows an assay comprising a Single Chamber system.Identifiers “A” and “B” represent non-FFL protein domains having wellcharacterized dimerization properties such as Glutathione S-transferase(GST) and glutathione or IgG FC-region and protein A.

FIG. 27A-B shows the schematic representation of sequence domains of thebiolumniogenic BoNT/A substrate and the principle of the bioluminescentassay. FIG. 27A shows FFL-L1 SL2TAH that was optimized for rapid BoNT/Acleavage (SEQ ID NO:25): “FFL” represents the full-length fireflyluciferase protein containing amino acid residues 1-550; “L1” representsLinker-1, a serine-glycine linker sequence containing four glycines, oneserine, and four glycines (G₄SG₄); “S” represents a cleavable portion ofSNAP25 containing amino acid residues 171-206; “L2” represents Linker-2,a glycine linker sequence containing six glycines; “T” represents anoptional positive control, a cleavable sequence containing amino acidresidues ENLYFQG recognized by the Tobacco Etch Virus protease; “A”represents a lysine anchor peptide containing eight to ten lysines andone glycine, one leucine, and one glutamic acid (K₈₋₁₀+GLE); and “H”represents a hexahistidine tag for protein purification (H₆). FIG. 27Bshows a schematic of the bead-based bioluminescent BoNT detection assay.The lysine-rich anchor sequences are marked with an anchor symbol andare positioned near the bead. BoNT-cleavable sequences (dark grey middlerectangle) are positioned between the anchor and the luciferasecomponents. The luciferase components are located at the end of thesubstrate, furthest from the bead (dark grey rectangle). When theBoNT-cleavable sequence is cleaved, the cleaved product is released fromthe bead and the bioluminescent signal can be detected.

FIG. 28A-B shows bioluminogenic BoNT substrates. (A) Recombinant fusionproteins FFL and FFLSH contain full-length luciferase (1-550 residues),Tev cleavage site and polyhistadine (H₆) tag. FFLSH contains a BoNT/Acleavable human SNAP25 residues 171-206 (SNAP). (B) BoNT/A cleavage ofFFL and FFLSH with and without subsequent addition of YCl₃. Theconcentration of BoNT/A was picomolar; Background=reaction buffer only;s/n=signal to noise.

FIG. 29A-C shows the testing of fire fly luciferase protein substratesfor BoNT detection. Scheme of the experiments with thehexahistidine-tagged substrate FFL-SNAP25-His₆. (FFLSH) (A); results ofthe control experiments (B); Cleavage assay with a BoNT/A LC dilutionseries (C). Supernatant only (left bars) and Ni-NTA beads (right bars)were tested. The concentration of FFLSH was 463 nM in all experiments.

FIG. 30(A) shows BoNT-mediated cleavage of the bioluminogenic substratein solution following binding on Ni-NTA beads. Performance of thesepharose bead-immobilized bioluminogenic substrate in human serumspiked with different BoNT serotypes is shown in FIG. 30(B).

FIG. 31 shows aggregate formation of histidine-tagged luminogenic BoNTsubstrate FFLSH with yttrium (III) ions. FFLSH and bovine serum albumin(BSA), 1 μg each, were passed through filters of different pore sizesand analyzed by SDS gel electrophoresis (left panel). In the presence ofYCl₃ (10 mM) FFLSH and FFLSH/BSA mixtures forms micro aggregates thatwere efficiently removed by filtering. BSA alone does not formaggregates with Y(III) ions (right panel). The 0.22 and 0.45 μm filtercontained low protein-binding PDVF membranes, whereas the 1.00 μm filtercontained glass fibers. Note that FFLSH but not BSA was already removedby the glass-containing filter in absence of Y(III) ions.

FIG. 32 shows BoNT/A ALISSA with luminogenic substrates. FFLSH (0.1 μg)was treated with ALISSA-bead immobilized BoNT/A complex from BoNTsamples of concentrations as indicated. The supernatant was subsequentlyincubated with Ni-NTA agarose beads. The Ni-NTA beads were collected onspin columns and both beads and flow through were tested for luciferaseactivity.

FIG. 33 shows development of the FFL inhibitor domain forBoNT-activatable luminogenic substrates. FFL inhibition assay usingequimolar solutions of FFL with ONE-Glo substrate (Promega), in presenceof an anti-FFL mAb (10F9H4), anti-BoNT/A IgG (control), serum from anFFL-immunize mouse (#58 mouse serum) and an antibody-free control (PBS).

FIG. 34 shows an exemplary BoNT/A ALISSA assay with sera and tissueextracts of intoxicated and non-intoxicated mice. Pairs of mice werei.p. injected with BoNT/A complex in the following amounts: (A) 200 pg;(B) 100 pg; (C) 20 pg; and (D) 0 pg (mock injection). Negative controlwas reaction buffer only (no serum or organ extracts).

FIG. 35 shows BoNT/A ALISSA detection of BoNT/A in mouse serum in anintoxication/rescue model. Groups of 4 mice were i.v.-injected with1,000 pg/mouse (143 mouse LD₅₀) of BoNT/A complex. 10 min later, micewere injected with a combination of BoNT/A neutralizing mAbs, 2.5 μgF1-2 (anti heavy chain) and 2.5 μg F1-40 (anti light chain),respectively. Blood was drawn at the indicated times after injection ofthe rescue antibody. 100 μL serum per animal was used for ALISSAdetection of BoNT/A. Controls received no BoNT/A. The horizontal barmarks the level of the background signal.

FIG. 36A-D shows ALISSA examples for BoNT/A, E, and anthrax LF. Resultsof the bead-free reaction (substrate and toxin only) were compared tothe bead-based ALISSA (see components) for dilution series of the toxinsin serum (a, c, d). ALISSA components were: rabbit polyclonal antibodiesto Clostridium botulinum A toxoid, protein NG beads, fluorogenic peptide(SNAPtide), and BoNT/A complex (a); Rabbit polyclonal antibodies againstBoNT/E, protein NG beads, fluorogenic substrate (SNAP Etide), and BoNT/Ecomplex (c); ALISSA with: goat anti-anthrax LF, protein A and G beadsand peptide substrate (MAPKKide), and LF (d); Standard curve of thefluorescence signal of unquenched calibration peptide (SNAPtide) inBoNT/A reaction buffer (b).

FIG. 37 shows the handling of the beaded ALISSA resin in spin columns.The spin column was loaded after the immunoprecipitation step using aLuer-Lock screw adaptor screw cap (a). Multiple sample resins werewashed by gravity flow (b).

FIG. 38 shows the dilution table used to generate a standard calibrationcurve for ALISSA.

FIG. 39 shows an exemplary ALISSA assay as performed to detect targetenzyme chitinase. Sera from 10 patients were tested. The patients areidentified as “#3”, “#8”, “#87”, “#37”, “#128”, “#22”, “#92”, “#90”,“#40”, and “#38”. Hatched bars correspond to CHIT1 serum ALISSA. Solidbars correspond to serum chitotriosidase activity. Positive control seraare indicated to the far left.

FIG. 40A-B shows an exemplary ALISSA assay as performed to detect targetenzyme Pep1 and Pep2 in patients with fungal infection (A) and no fungalinfection (B). Patient sera are identified as “#3”, “#12”, “#37”, “#87”,“#128”, “#22”, “#90”, “#38”, “#40” and “#92”. Hatched bars correspond toPep1 results. Solid bars correspond to Pep2.

FIG. 41 shows the experimental scheme for the ALISSA detection of BoNT/Ain intoxicated mouse sera using a bead-based immunomatrix. Thebead-based immunomatrix was prepared using the monoclonal 5A20.4 (antiBoNT/A light chain) antibody (provided by Dr. James Marks, University ofCalifornia, San Francisco) coupled to porous beads. An experiment wasperformed using BoNT/A cleavable peptide #115 (SEQ ID NO:21) to testsera with and without anti-body coupled beads (bead-based and bead-free,respectively). The bead-based ALISSA demonstrated superior sensitivitycompared with the bead-free experiment.

FIG. 42 shows the steps involved in the ALISSA detection of BoNT/A inintoxicated mouse sera. Step 1 involves preparation of the immunomatrixby coupling the monoclonal 5A20.4 (anti BoNT/A LC) antibody to porousbeads. Step 2 involves immunocapturing of the BoNT/A complex present inthe serum from intoxicated mice. Step 3 involves the enzymatic reactionused to detect BoNT/A cleavage of fluorescent peptides. BoNT/A cleavablepeptide #115 (SEQ ID NO: 21) and control peptide #113 (SEQ ID NO: 5)were tested simultaneously (#115 contains a 5-FAM at the N-terminus and#113 contains a 4-MU at the N-terminus), while cleavable peptide #116(SEQ ID NO: 22) and control peptide #112 (SEQ ID NO: 19) were testedsimultaneously (#116 contains a 4-MU at the N-terminus and #113 containsa 5-FAM at the N-terminus) in the same ALISSAs.

FIG. 43A-B shows the standard curves for the ALISSA with BoNT/A1complex-spiked pooled mouse serum. FIG. A shows the standard curve forthe 5-FAM labeled substrate (#115) and FIG. B shows the standard curvefor the 4-MU labeled substrate (#116).

FIG. 44 shows the experimental scheme for the mouse model of botulism.Mice were either administered BoNT/A complex through intravenous (i.v.)injection or intragastric (i.g.) gavage. Tail blood was drawn at theindicated time and BoNT/A ALISSA was performed using mouse serum. Micein Group I were administered i.v. injections containing differentconcentrations of BoNT/A complex (0, 4 pg, 20 pg, and 100 pg BoNT/Acomplex per mouse); serum was tested for BoNT/A using ALISSA after 1hour. Mice in Group II were administered i.v. injections containing 100pg BoNT/A complex per mouse; serum was tested for BoNT/A using ALISSAafter 1, 3, 5, and 7 hours. Mice in Group III were administered 4 μgBoNT/A complex through i.g. gavage and serum was tested for BoNT/A usingALISSA after 2, 5, and 7 hours.

FIG. 45A-C shows quantification of BoNT/A serum of i.v. intoxicated mice(n=4 per group). After 1 hour, the sera from Group I mice were testedusing the BoNT/A cleavable substrates (#115 (SEQ ID NO: 21) and #116(SEQ ID NO: 22) (FIG. 45 A). The concentration of BoNT/A was determinedwith BoNT/A cleavable substrates, #115 (SEQ ID NO: 21) and #116 (SEQ IDNO: 22), from the ALISSA standard curves generated with BoNT/A complexserially diluted with normal mouse serum. Linear correlation equationswere used to convert the fluorescence counts obtained from non-BoNT/Aspecific cleavable of control substrates, #112 (SEQ ID NO: 19) and #113(SEQ ID NO: 5). FIG. 45 B shows a bar graph depicting the results fromFIG. 45 A. 5-FAM labeled BoNT/A cleavable substrate (#115) combined with4-MU labeled control peptide (#113) results are shown as the white bar,and 4-MU labeled BoNT/A cleavable substrate (#116) combined with 5-FAMlabeled control peptide (#112) results are shown as the grey bar. FIG.45 C shows a line graph plotting the BoNT/A concentration detected overtime (sera was tested at 1, 3, 5, and 7 hours post intoxication. BoNT/Aserum concentrations for each time point were calculated by dividing theresults of the cleavable peptide by the results of the control peptide.

FIG. 46A-C shows the BoNT/A ALISSA results using sera from mice thatwere administered BoNT/A through i.g. gavage. BoNT/A cleavablesubstrates, #115 (SEQ ID NO: 21) and #116 (SEQ ID NO: 22), were used todetect the presence of BoNT/A and substrates, #112 (SEQ ID NO: 19) and#113 (SEQ ID NO: 5), were used as negative controls. Mice received 4 μgBoNT/A complex and serum was tested 2, 5, and 7 hours after delivery(FIG. 46 A). The bar graph in FIG. 46 B illustrates that time isrequired for BoNT/A toxin perfusion into the blood stream. FIG. 46 Cshows the results from mice that received 1 μg BoNT/A through i.g.gavage. The tail blood of mice was tested at 2, 5, 7, 8, 24, and 48hours after toxin delivery.

FIG. 47 shows the experimental protocol for the ALISSA quantification ofintraneuronal BoNT/A1.

FIG. 48A-B shows ALISSA standards for BoNT/A quantification. FIG. 48 Ashows standards using 20 nM recombinant BoNT light chain (white bar) orBoNT/A1 complex in high potassium (K⁺) medium. FIG. 48B shows standardsfor ALISSA with cell intoxication (white bar) and wash media (black bar)in a high K⁺ buffer. Control values are subtracted from total values.

FIG. 49A-C shows the ALISSA standards and quantification of BoNT/A1detected in extracts, pellets, supernatants and wash buffers ofintoxicated rat primary hippocampus neurons. A standard curve wasobtained with the 150-kDa holotoxin spiked into cell lysis buffer (A).Initial experiments detected the presence of toxin in both cell extractand cell pellet (B, black bar and white bar, respectively).Quantification results from ALISSA in extracts (left bar graph),supernatants (middle bar graph), and wash buffer (right bar graph) (C).Different concentrations of BoNT/A1 holotoxin were added to the mediumused for cell intoxication (control, no BoNT (white bar); 20 nM BoNT/A1(black bar); 2 nM BoNT/A1 (grey bar)). At 2 nM BoNT concentration, thecells retained 0.27 fmol BoNT (2,167 active BoNT molecules per cell).The BoNT/A1 concentrations were measured in a final sample volume of 1mL.

FIG. 50 shows the results from the first BoNT/A ALISSA with sera fromadult botulism patients. Sera from uninfected adults (left bar graph),adult patients infected with food-borne botulism (middle bar graph), oradult patients infected with wound botulism (right bar graph) weretested using ALISSA. The BoNT/A cleavable substrate, #115 ((SEQ ID NO:21), left bar in each pair), and the non-cleavable control peptide, #112((SEQ ID NO: 19), right bar in each pair), were tested. Adult sera wereprovided by the California Department of Public Health.

FIG. 51 A-B shows the results from a BoNT/A ALISSA with infant botulismpatient serum. Pooled human serum (negative control, left bar graph) andserum from infants infected with botulism (right bar graph) was testedusing ALISSA (FIG. A). The BoNT/A cleavable substrate, #115 ((SEQ ID NO:21), left bar in each pair), and the non-cleavable control peptide, #112(SEQ ID NO: 19), right bar in each pair), were tested in the assay.Infant sera were provided by the California Department of Public Health.FIG. B shows the BoNT/A ALISSA standard curve for the assay that allowsconversion of RFU into the concentration of BoNT/A holotoxin.

FIG. 52A-B shows screening of anti-BoNT/A camelid antibodies (VHHs) forBoNT ALISSA detection in BoNT-spiked pooled human serum. (A) The VHHsJDY-33, JDY-46, JEP-2, and JFK-2 are directed against BoNT LC. 5A20.4 isa monoclonal humanized anti-BoNT/A LC antibody from Dr James Marks ofUCSF. (B) BoNT/A ALISSA with a bivalent heterodimer VHH that consists ofa fusion of thioredoxin (Trx), an E-tag (E), the H7 or C2 VHH, and alysine decamer at their C-terminus (10K).

FIG. 53A-C shows BoNT ALISSA with VHH hetero-dimers and trimers. (A)Comparison of BoNT-specific heterodimer (JIA-44) versus heterotrimer(JIA-31) VHHs in bead-based ALISSA; and (B, C) Surface Plasmon Resonance(SPR) kinetic analysis of the heterodimer in (B) single-cycle and (C)multi-cycle mode. The dissociation constant K_(D) of the heterodimer VHHJIA-44 was determined to be 64.4 to 65.8 pM.

FIG. 54A-C shows affinity maturation of VHH domains of single chainalpaca antibodies, displayed on S. cerevisiae cells. FACS analysis of(A) H7 mutant and (B) the H7 wildtype (WT). The oval in each upper rightquadrant in (A and B) marks the gate used to collectLCa-FITC^(high)/C-Myc^(high) cells. (C) Photography of a 96-wellmicrotiter plate with colonies from selected individual yeast cellsgated in (A), which are expected to express mutated H7 with a BoNT/A LCbinding affinity that is significantly higher than that of the WT. Thedark dots in well centers are the living colonies (n=63).

FIG. 55 shows screening of BoNT/B VHHs for best performance in abead-based BoNT/B ALISSA. The three letter naming scheme (starting withJ) was adopted form Dr. Shoemaker who isolated these VHHs.

FIG. 56A-B shows the ALISSA using pipette tips columns containingaffinity microcolumns. FIG. 56(A) shows a schematic of the ALISSA usingpipette tips containing affinity microcolumns. The biological sample wasadded to an affinity pipette tip containing the bead-based immunomatrix(immobilized BoNT/A antibody). Flow-through was washed away while BoNT/Awas bound by the BoNT/A antibody. BoNT/A specific fluorogenic substrateswere added to the affinity pipette tip. Unquenched fluorophore wasreleased upon substrate cleavage. Fluorescence of cleaved substrate wasread using a waveguide sensor. FIG. 56B, left panel, shows the affinitypipette tip columns containing microcolumns that can be used inconjunction with an electronic multichannel pipettor. FIG. 56B, rightpanel, shows the affinity tips fitted onto a Versette automatedworkstation.

FIG. 57A-F shows BoNT/A ALISSAs using microcolumns. (A) A dilutionseries was performed using pooled human serum spiked with increasingconcentrations of BoNT/A light chain (LCa). Microcolumns were used withan immunomatrix containing BoNT/A specific antibodies (5A20.4).Increasing cleavage of the BoNT/A cleavable substrate is seen withincreasing concentrations of BoNT/A, demonstrating that BoNT/A lightchains cleave the BoNT/A cleavable substrate. (B) A similar experimentas seen in (A) was performed using affinity microcolumns with animmunomatrix containing antibodies specific to BoNT/B light chains(anti-LCB monoclonal (6B.1)). A dilution series was performed usingpooled human serum spiked with increasing concentrations of BoNT/A lightchain. Negligible cleavage of the 5-FAM BoNT/A cleavable substrate(#201a) was seen, indicating that BoNT/A does not specifically bind amicrocolumn immunomatrix containing BoNT/B antibodies. (C) An ALISSAusing microcolumns with an immunomatrix containing the BoNT/A VHH H7antibody was performed; however, no toxin was added to the microcolumnin this experiment. Each lane represents data from one affinitymicrocolumn pipette tip. (D) ALISSA with microcolumns was performedusing an immunomatrix containing no conjugated antibody. Pooled humanserum spiked with 10 pM BoNT/A light chains (dark-grey bars,underivatized tips 1-6) and no toxin (light-grey bars, underivatizedtips 7-12) was added to the microcolumn. There was no difference incleavage of the 5-FAM labelled BoNT/A cleavable substrate (#201a)between those microcolumns treated with BoNT/A and those treated with noBoNT/A. These results indicate that BoNT/A does not bind theimmunomatrix non-specifically. Each figure represents the data obtainedfrom one single affinity tip containing a microcolumn. (E) ALISSA withmicrocolumns was performed using an immunomatrix containing the BoNT/Aspecific antibody, VHH H7. Microcolumns were either treated with pooledhuman serum containing 10 pM of BoNT/A or were not treated with BoNT/A.There was no difference in cleavage of 5-FAM labelled BoNT/A controlpeptide (204a) between the microcolumns treated with BoNT/A and themicrocolumns that were not treated with BoNT/A. These results indicatethat BoNT/A does not cleave the control peptide. Each figure representsthe data obtained from one single affinity tip containing a microcolumn.(F) Results from an automated BoNT/A ALISSA with microcolumns on aVersette liquid handling workstation. The samples were automaticallyprocessed on microcolumns loaded with Dr. James Marks' monoclonalantibody 5A20.4. Nine hundred aspirate/dispense cycles were used persample (in triplicates).

FIG. 58 shows the nucleic acid sequence of FFL1-478SNAP25FFL265-550 (SEQID NO:3). In this embodiment of an overlapping split FFL having a SNAP25sequence insert, nucleic acids encoding amino acids 1 through 478 of FFLis included in the first FFL segment. The corresponding SNAP25 sequenceswith the BoNT/A cleavage site are indicated in bold and underlined text.The hexahistidine tag is indicated in bold and italic text.

FIG. 59 shows the amino acid sequence of FFL1-478SNAP25FFL265-550 (SEQID NO:4). In this embodiment of an overlapping split FFL having a SNAP25sequence insert, amino acids 1 through 478 of FFL is included in thefirst FFL segment. The corresponding SNAP25 sequences with the BoNT/Acleavage site are indicated in bold and underlined text. Thehexahistidine tag is indicated in bold and italic text.

FIG. 60 shows the complete amino acid sequence of human SNAP25 (SEQ IDNO: 11; Swissprot Accession #P60880). The BoNT/A cleavage andrecognition site (SEQ ID NO: 2) is indicated in bold and underlinedtext.

FIG. 61 shows the complete amino acid sequence of “FFL-L1SL2TAH”:FFL[1-550] L1 [G₄SG₄] SNAP25[171-206] L2[G₆] T[ENLYFQG]A[K₈₋₁₀+GLE]-histag (SEQ ID NO:25). The full-length firefly luciferasesequence (amino acid residues 1-550) is indicated in normal text, theglycine/serine linkers are indicated in underlined text, the SNAP25sequence (amino acid residues 171-206) is indicated in italicized text,the TEV protease cleavage site sequence is indicated in bold text, thelysine-rich anchor sequence is indicated in bolded and underlined text,and the polyhistadine tag is indicated in bolded and italicized text.

FIG. 62 shows the complete amino acid sequence of full length fireflyluciferase from Photinus pyralis (SEQ ID: NO 26).

FIG. 63 shows the amino acid sequences of peptides Octa-lysine AnchorPeptide “Anchor (K₈+GLE)” (SEQ ID NO: 30), Deca-lysine Anchor Peptide“Anchor (K₁₀+GLE)” (SEQ ID NO: 31), Polyhistadine Tag “Histidine Tag(H₆)” (SEQ ID NO: 32), Tobacco Etch Virus Protease Cleavage Site (SEQ IDNO: 33), Serine-Glycine Linker 1 “Linker 1 (G₄SG₄)” (SEQ ID NO: 34), andGlycine Linker 2 “Linker 2(G₆)” (SEQ ID NO: 35).

DETAILED DESCRIPTION

The following description of the invention is merely intended toillustrate various embodiments of the invention. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to one skilled in the artthat various equivalents, changes, and modifications may be made withoutdeparting from the scope of the invention, and it is understood thatsuch equivalent embodiments are to be included herein.

Provided herein is a laboratory method for detecting the activity of atoxin or enzyme that utilizes common lab equipment and commerciallyavailable reagents, and is therefore expected to be reproducible by anyreasonably well equipped biological laboratory. This method is referredto herein as an Assay with a Large Immuno-sorbent Surface Area (ALISSA).The examples set forth herein provide a detailed protocol for theALISSA, as well as an analysis of the effect of various experimentalparameters on the assay. In certain embodiments, the ALISSA is employedfor the detection of botulinum toxin A (BoNT/A). For example, theexemplary experimental results disclosed herein show that the assay candetect less than 0.5 fg of BoNT/A holotoxin in 1 mL serum, milk, orGP-diluent. Based on these results, the ALISSA is at least about32,000-fold more sensitive than the life mouse assay and about160,000-fold more sensitive than the Enzyme-linked Immunosorbent Assay(ELISA). In certain embodiments, the turnaround time for the ALISSA isone to two hours, which is significantly faster than the life mouseassay (˜48 hours) and faster than ELISA (˜3 hours). The exemplaryexperimental results obtained herein were obtained with BoNT type A(BoNT/A), but could be applied just as easily to other BoNT serotypes orother toxins as well as enzymes.

The ALISSA may be used in combination with or in place of the mouse LD₅₀assay for measuring the potency of products containing BoNT. Owing toits remarkable sensitivity and rapid turnaround time, the ALISSAprovides a beneficial value compared with the “gold standard” mouseassay. Additionally, use of the ALISSA may provide a cheaper method oftesting BoNT products as it may result in a reduction of the number ofsamples or subjects required for testing. The methods and kits providedherein may be used to evaluate the potency, stability, and efficacy ofproducts containing BoNT. The methods and kits may also be used toevaluate products that contain, but are not limited to, activeingredients such as onabotulinumtoxinA, rimabotulinumtoxinB,abobotulinumtoxinA and incobotulinumtoxinA to measure the functionalactivity of the toxin protein. In some aspects, the method herein may beused to confirm the amount of active ingredient contained in theproduct. The method provided herein may also be used as ahigh-throughput assay for testing large quantities of samples.

The ALISSA avoids interference with other sample components by using ahighly specific affinity matrix and exploiting the natural catalyticactivity of the toxin or enzyme (“target”) with a target-specificsubstrate. Both of these steps amplify the signal via localizedenrichment of the toxin and enzymatic conversion of multiple substratemolecules per toxin molecule. In certain embodiments, ALISSA comprisestwo main steps: 1) binding and enrichment of toxin or enzyme on abead-based enrichment matrix and removal of unspecific bindingmolecules, and 2) determination of the enzymatic activity of theimmobilized toxin or enzyme based on cleavage of a specific fluorogenicor bioluminescent substrate. The enrichment matrix may compriseprotein-A, protein-G, or protein A/G conjugated sepharose, agarose, ormagnetic beads coupled and cross-linked to anti-toxin, anti-enzyme, oranti-toxin substrate antibodies. For example, the enrichment matrix maycomprise protein-A conjugated sepharose beads coupled and cross-linkedto anti-BoNT antibodies. Additionally, in other aspects the beads mayalso be coupled and cross linked to anti-toxin substrate antibodies. Theanti-toxin substrate antibodies may bind a portion of the substrateincluding the fluorophore, the quencher, or amino acids from the SNAP-25sequence. For example, the enrichment matrix may comprise protein-A/Gconjugated agarose beads coupled and cross linked to anti-FITCantibodies that bind the fluorescent 5-FAM label conjugated to the BoNTsubstrate. In another example, the enrichment matrix comprisesprotein-NG conjugated agarose beads coupled and cross liked toanti-DABYCL antibodies that bind the DABYCL conjugated to the BoNTsubstrate. In yet another example, the enrichment matrix comprisesprotein-NG conjugated agarose beads coupled and cross linked toantibodies that bind a portion of the SNAP-25 sequence. Furthermore, theenrichment matrix may comprise a mixture of both anti-toxin antibodiesand anti-toxin substrate antibodies. In other aspects the enrichmentmatrix comprises cyanogen-bromide (CNBr) activated Sepharose beadscoupled and cross linked to anchor (lysine rich) sequences fused to atoxin substrate. For example, the enrichment matrix may compriseCNBr-activated Sepharose beads coupled and cross linked to lysine richsequences fused to a luminescent BoNT substrate. In other aspects, theenrichment matrix may comprise nickel nitrilotriacetic acid (Ni-NTA)beads coupled and cross linked to affinity tags fused to luminogenicBoNT cleavable fusion proteins. The immunosorbent support providedherein can be comprised of either loose beads or one or more fixedcolumns.

As used herein, the term “target” when used to refer to a toxin orenzyme, is used to refer to any chemical, biochemical or biologicalspecies or compound that is known or referred to in the art as a toxinor an enzyme. A target toxin or target enzyme includes those compoundshaving proteolytic, catalytic or enzymatic activity. A target toxin ortarget enzyme includes those compounds able to modify a substrate so asto alter or change the substrate's chemical structure or apparentstructure or activity. For example, a botulinum neurotoxin type A is a“target” toxin that has proteolytic activity and is able to cleave itsspecific substrates. For all botulinum serotypes this may include theBoNT complex, holotoxin, and light chains. As another example, achitinase is a “target” enzyme that has enzymatic activity. In anotherexample, anthrax lethal factor is a “target” enzyme that has enzymaticactivity.

As used herein, the term “substrate” is used to refer to any chemical,biochemical or biological species or compound that complexes with,reacts with, is capable of being modified by, or otherwise interactswith a toxin or enzyme having bioactivity. For example, a botulinum typetoxin is a protease able to enzymatically cleave specific proteinsubstrates such as synaptic membrane proteins, SNARE proteins or SNAP-25proteins. As another example, a chitinase substrate interacts with achitinase enzyme such as endochitinase or exochitinase.

As used herein, the term “fluorogenic substrate”, and “fluorophore” maybe used interchangeably to describe a substrate that is hydrolyzed by orotherwise reacted with a target toxin upon contact therewith, producinga complex, product or other derivative thereof which liberatesfluorescence upon excitation by a suitable light source.

As used herein the term “bioluminescent substrate”, “luminescentsubstrate”, and “luminogenic” protein may be used interchangeably todescribe a substrate that is activated by or otherwise interacts orreacts with a target toxin upon contact therewith, producing a complex,product, or other derivative thereof which emits light at distinctwavelengths suitable for detection as desired.

As used herein the term “homologous” is used to refer to any nucleicacid or protein sequence that displays at least 90% similarity with anamino acid sequence wherein the resulting protein still retains itsdesired functional properties.

In accordance with the method of the ALISSA, one or more samples from asource suspected of containing a toxin is obtained and then contactedwith a substrate composition comprising a toxin substrate, such as afluorogenic or luminogenic substrate or a mixture thereof, for a periodof time and under conditions sufficient to permit the toxin to reactwith the toxin substrate to cause a measurable change in a property suchas fluorescence or light emission, or the resulting reaction product.

In general, the toxin or enzyme contained in the sample is first boundon an enrichment matrix such as a bead-based immuno-affinity matrixcontaining immobilized anti-toxin specific antibodies. Immobilization ofthe antibodies to the matrix can be by a variety of methods, including,for example by covalent crosslinking of the Fc region of the antibody tothe beads. Once bound, the toxin or enzyme molecules retain enzymaticfunction and specificity for its substrate.

The natural substrate of BoNT/A is the 25-kDa synaptosomal-associatedprotein (SNAP 25), which it cleaves at distinct sites, therebypreventing the release of neurotransmitters (Schiavo 1993a; Schiavo1993b). In those embodiments wherein BoNT/A is being detected, theenzymatic activity of BoNT/A may be utilized to cleave the fluorogenicsubstrate SNAPtide, which is a synthetic, commercially available,13-amino acid peptide that contains the native SNAP-25 cleavage site forBoNT/A (U.S. Pat. No. 6,504,006). In those embodiments wherein a BoNTtype other than type A is being detected, the fluorogenic substrate maybe any substrate that is specifically cleaved by the BoNT type beingdetected. For example, if BoNT B, C, D, F or G is the toxin beingdetected, then the substrate comprises a fragment of vesicle-associatedmembrane protein (VAMP) containing the respective BoNT cleavage site,and if BoNT C or E is the BoNT toxin, then the toxin substrate comprisesa fragment of SNAP-25 containing the respective BoNT cleavage site. Inthose embodiments where a different class or type of toxin other than aBoNT is being detected, the substrate may be any substrate that isspecifically cleaved or catalyzed by the toxin being detected.

One aspect of the ALISSA provides a method for detecting toxin or enzymewhich avoids interference with other sample components by use of hightoxin-specific affinity matrix and toxin-specific substrates. Forexample, use of a high affinity BoNT/A enrichment matrix and aBoNT/A-specific substrate reduces or avoids interference by othercomponents present within a sample thus amplifying the signal andincreasing the assay's sensitivity. Use of a toxin-specific substratealso exploits the natural proteolytic activity of the toxin. Signalamplification is achieved by localized enrichment of the toxin andthrough enzymatic conversion of substrate molecules.

In certain embodiments, the capture matrix is designed to stably enrichthe toxin while retaining enzymatic activity. The capture matrix mayalso purify toxin from non-specific components or proteases presentwithin the sample. Use of a beaded protein A matrix to bind anti-toxinantibodies via FC-region allows orientation of the antibody bindingdomains away from the bead surface and into the surrounding fluid. Thisaugments and provides increased accessibility for toxin molecules. Useof a bead-based assay also allows for wash steps that diminishinterference by other proteases. The ALISSA provides a considerablyfaster and more sensitive method for detecting toxin and its activity.In certain embodiments, the capture matrix is designed to stably enrichthe toxin and the toxin substrate while retaining enzymatic activity. Inother embodiments the enrichment matrix is a double affinity matrixcomposed of one antibody directed to the target toxin and anotherantibody directed to the toxin substrate. This bivalent immobilizationmatrix results in further enhancement of enzyme catalysis by providing astrong substrate-bead interaction in combination with a strongenzyme-bead interaction. The antibody directed to the toxin substratemay bind the fluorogenic label conjugated to the substrate, the darkquencher conjugated to the substrate, or the amino acid structure of theSNAP-25 peptide.

The ALISSA may also be used with one or more columns for high analyticalspecificity and detection of toxin or enzyme in complex biologicalsamples. This column-based ALISSA method may be used for specificallybinding the target toxin and removing unspecific binding molecules. Thecolumn may be packed with a bead-based enrichment matrix that containsbound ligands, which specifically bind the target toxin or enzyme.Briefly, the sample containing the target toxin may be added to thecolumn containing the ligand-bound enrichment matrix. The sample may beintroduced to the column via gravity flow or pressure. Additionally, thesample may be incubated with the enrichment matrix to allow theimmobilized ligand to bind the target toxin. The sample may then beremoved, leaving the target toxin bound to the immobilized ligand on theenrichment matrix within the column while the non-specific molecules maybe washed away. Next, a toxin substrate capable of eliciting adetectable signal may be added to the column containing the targettoxins bound to the enrichment matrix. Upon substrate cleavage, thedetectable signal may be measured. In some examples, the signal may bedetected after elution of the sample containing the detectable signal.In certain examples, the toxin substrate may be either a fluorogenicpeptide or a luminogenic peptide. In some embodiments, the column usedin the ALISSA is an affinity microcolumn containing an enrichment matrixcomprised of an immuno-affinity matrix.

Any variety of one or more commercialized columns may be used for thecolumn-based ALISSA including, but not limited to, gravity-flow columns,spin columns, and pressure columns. In some embodiments, two or morecolumns may be used for the ALISSA; however, one column is the preferredmethod of use. Additionally, pipette tip columns containing affinitymicrocolumns mounted into pipette tips may be used for the column-basedALISSA. In some examples, the pipette tips may be disposable. Forexample, the affinity microcolumns that may be used in the column-basedALISSA may be dextran glass columns from Intrinsic Bioprobes, Inc.(acquired by Thermo Fischer Scientific) and may contain microcolumnsmounted into pipette tips. The affinity microcolumns mounted intopipette tips may be used as described in U.S. Pat. No. 7,087,164 B2. Insome examples the microcolumns mounted into pipette tips may be used inconjunction with an inexpensive, robust automated high-throughput methodfor detecting BoNT in biological samples. For example, a microcolumnrobotic pipetting workstation system may be used for the automatedmicrocolumn based BoNT detection system for use as a high-throughputsystem. The system may be programmed to conduct automated functions suchas microcolumn-enrichment of BoNT/A from spiked samples of human serumand microcolumn washes. In other examples, the microcolumns mounted intopipette tips may be used in conjugation with an electronic multichannelpipettor. In certain aspects this exemplary method of detecting BoNT/Amay also be used to detect other BoNT serotypes such as B, C, E, and Fas well and a wide variety of other toxins or enzymes.

In certain embodiments, antibodies may be conjugated to the enrichmentmatrix of the affinity microcolumns. For example, the BoNT/A light chainspecific monoclonal antibody, 5A20.A, may be conjugated to beads to bindthe LC of BoNT/A. However, other affinity reagents may be used formicrocolumns such as fusion proteins that contain antigen-bindingfragments like scFvs or the binding domains of single chain antibodies.Additionally, recombinant alpaca single chain antibodies (VHH) may beused in the bead-based microcolumn ALISSA for detection of BoNT. Toimprove the performance of ALISSA, bivalent and multivalent VHHs may beused to bind BoNT. Cyanogen bromide-activated Sepharose beads may beused to coupled VHHs or antibodies to ALISSA beads. In other examplesthe affinity microcolumns may be optimized to contain VHH proteinsgenerated from affinity maturation of BoNT/A and BoNT/B specific VHHreagents using a yeast display technique. In some aspects, the toxinspecific antibodies are immobilized using an immuno-affinity matrixcontaining protein NG-coated beads. In other examples, the antibodiesmay readily be utilized for the ALISSA after direct immobilization viaamine groups to Cyanogen-bromide activated Sepharose beads. The ALISSAmay improve the diagnosis of botulism and other toxins significantly andmay serve to protect humans in biomedical and bio-defense scenarios. Themethod may also be applied for the routine testing of foods and forforensic investigation.

In certain embodiments, the detailed steps that are necessary for usingALISSA with a bead-based immuno-affinity matrix have been provided. Forexample, an immunocapture method may be used to bind BoNT toxinsincluding BoNT complexes, holoenzymes, and light chains for use indetecting BoNT. Different antibodies recognizing different epitopes onBoNT may be used to conjugate to the immuno-affinity matrix. Forexample, antibodies that recognize BoNT/A and BoNT/B (camelid heavychain antibodies), or BoNT/A light chains (BoNT/A LC) (5A20.4 monoclonalantibody), and BoNT/B LCs (2B24) may be coupled to CNBr-activatedSepharose beads for use in the bead-based ALISSA. These conjugated beadsmay then be used to bind BoNT complexes, holoenzymes, and/or lightchains. BoNT cleavable substrates may then be used to detect BoNTactivity as described.

Detection of all BoNT serotypes including subtypes may also achievedutilizing novel fluorogenic or luminogenic substrates. The botulinumneurotoxins cleave a variety of vertebral SNARE (Soluble NSF attachmentprotein receptor) in vivo and in vitro. While some fluorogenic BoNTsubstrates based on natural SNARE sequence are known (Schmidt 2003), thepossible interference by sterically demanding fluorophore or quenchermoieties on the catalytic cleavage reaction of such fluorogenic peptidesremains a concern. Novel substrates that achieve higher chemicalstability and comparable or superior sensitivity as compared to priorpeptides have been provided. Preferably, fluorophore substrates thatallow for efficient cleavable fluorophore and quencher combinations areselected for use in the ALISSA assay. Generally, fluorophore andquencher require proximities of about 10 nm or less to allow sufficientFRET-mediated quenching. Closer distances are also preferred to reducebackground fluorescence from the quenched substrates. In certainembodiments, use of bioluminescent substrates to allow for luminescentBoNT detection may be desired. Luminescent based assays can reduce oromit the requirement for a light source and provide greatersignal-to-noise ratios. Bioluminescent light in particular, can bedetected using less complex means such as with miniaturizedphotomultipliers or microscopic avalanche photodiodes. Furthermore,potential interference from background fluorescence due to inertcomponents of a microfluidic device is alleviated.

Novel fluorogenic substrates for BoNT serotypes such as serotypes A to Gare designed through use of peptide libraries having proteinogenic andas well as non-proteinogenic amino acids. Preferably, those substrateshaving resistance to non-BoNT proteases are selected for use with theALISSA or other immobilized antibody matrix based assay. Morepreferably, substrates designed so as to be more specifically andreadily cleaved by BoNT are also provided. Thus, the ALISSA may beuseful for detecting BoNT of all serotypes and subtypes in one or morebiological sample, in vitro or in vivo, using affinity capture of BoNTon microscopic beads coated with antibodies specific to the toxin. Theantibody-bound toxin retains its metalloprotease activity. In someaspects, the antibody on the beads is specific to the toxin substrateand binds the toxin substrate on the beads. In some aspects the beadscontain a mixture of antibodies directed to the toxin and the toxinsubstrate. The method includes use of a reporter molecule such as afluorogenic or bioluminescent substrate that is cleavable by one or moremolecules of the bound BoNT. Fluorescence is then detected using ahandheld ultraviolet (UV) light, a fluorescence excitation and/ordetecting tool, device or any suitable commercially availablefluorometer. In some embodiments the ALISSA may also be used to measurethe activity of other zinc metalloprotease enzymes.

In certain embodiments, methods are provided for identifying novelfluorogenic and luminogenic substrates that are useful for detecting thepresence and/or activity of a toxin or enzyme. Such toxin-specificsubstrates are useful for detecting, identifying and/or assaying for thepresence or activity of a toxin or enzyme in a sample at attomolarlevels of sensitivity. For example, it is known that botulinumneurotoxins cleave a variety of SNARE proteins. Sequences of naturalSNARE proteins have been used to produce fluorogenic BoNT peptidesubstrates. Such methods generally entail use of terminal fluorophoreand quencher molecule pairing (fluorescence through resonance energytransfer), or FRET moieties. It is difficult, however, to predict theeffect that sterically demanding fluorophore or quencher moieties willhave on the ability of the toxin to effectively cleave the resultingfluorophore modified substrate molecule. Some embodiments provide novelfluorogenic substrate peptides by employing synthetic peptide librariesto screen for those substrates that readily contain fluorophore andquencher combinations.

Cleavable fluorogenic substrates may be designed and used to detect thepresence of BoNT. In some aspects, a cleavable fluorogenic peptide,containing portions of the human SNAP-25 C-terminal sequence with anorleucine corresponding to residue M202, may be used to detect BoNT/Acleavage. For example, the norleucine may be located at the C-terminusof the peptide. A cleavable peptide may be labeled with at least onefluorogenic label conjugated at or near the N-terminus and at least onedark quencher may be conjugated at or near the C-terminus of thesubstrate. As used herein, the term “near the N-terminus” is used torefer to any position on the substrate that is within 5 amino acids ofthe N-terminus of the substrate, while the term “near the C-terminus” isused to refer to any position on the substrate that is within 5 aminoacids of the C-terminus of the substrate. The fluorogenic label may beconjugated to the substrate via a peptide bond, which enhances thestability of the substrate. Additionally, the substrates may containarginines of the L-isomeric form. In some embodiments a novelfluorogenic peptide containing the BoNT/B cleavable sequence ofvesicle-associated membrane protein (VAMP) may be used to detect BoNT/Bcleavage. In other aspects a fluorogenic cleavable peptide BoNT/Epeptide may be used to detect the presence of BoNT/E. Additionally, afluorogenic cleavable and control substrate may be designed and used inthe ALISSA to detect all BoNT serotypes, A to G. Internal controls mayalso be used for an endopeptidase-based BoNT detection assay todetermine the specificity of the BoNT ALISSA and the extent ofnon-specific proteases in a sample. In some aspects the control peptidescan be used in combination with the BoNT peptide substrates in the samesample. For example, control peptides labelled with an N-terminal5-carboxyfluorescein (5-FAM)—can be used with a BoNT cleavable substratecontaining a 4-Methylumbelliferone (4-MU) label and visa versa.

In a separate embodiment, methods are provided for the identification ofnovel luminogenic protein substrates. Using recombinant methodology,genetically engineered variants of recombinant luciferase proteins thatbecome activated by specific BoNT cleavage reactions are provided. Thus,the methods provide luminescent substrates specific for all serotypesand subtypes of botulinum toxin. Luminescence is detected using anysuitable commercially available luminometer.

Employed are two general approaches to exploiting bioluminescence foridentifying novel bioactive luminogenic substrates. The first includesuse of complementation of inactive luciferase fragments to restoreactive luciferase molecules. The second includes use of specificreactions that release D-luciferin as a substrate for firefly luciferase(FFL from Photinus pyralis). Complementation assays for luciferase aredescribed by Paulmurugan et al., “Combinatorial library screening fordeveloping an improved split-firefly luciferase fragment-assistedcomplementation system for studying protein-protein interactions,” AnalChem., 79:2346-2353 (2007); and Paulmurugan et al., “Firefly luciferaseenzyme fragment complementation for imaging in cells and livinganimals,” Anal. Chem. 77:1295-1302 (2005). Using describedcomplementation assays, split luciferase constructs are designed for usein detecting the presence of specific enzymatic activity. For example,an inactive N-terminal FFL fragment may be fused to a binding domain ofa protein having a known binding affinity for another binding partner.The other protein binding partner may be fused to the C-terminal portionof FFL and to a BoNT/A cleavable SNAP25 sequence, which is furtherimmobilized onto beads. Whereas such constructs are inactive when intheir fused, non cleaved state, upon interacting with a target toxinsuch as BoNT, the proteolytic activity of the toxin cleaves and releasesthe C-terminal portion of FFL. Upon release and binding of the bindingprotein domains, the FFL fragments recombine and a detectableluminescent signal is emitted due to restored FFL activity. An exampleof a protein containing a binding domain with a known binding affinityfor another binding partner would be glutathione-S transferase, whichhas a known binding affinity for a separate glutathione-S transferasemolecule.

The luminogenic substrates have the advantage that they can be producedinexpensively and in large quantities from cultures of engineered E.coli bacteria. Furthermore, luminogenic substrates reduce therequirements for expensive instrumentation. Simple and very sensitiveand even portable luminometers can be used instead of much moreexpensive fluorometric instrumentation. In certain embodiments,additional fusion proteins with the optimized construct of FFL, linkers,and SNAP-25 may be constructed by adding a green fluorescent protein toits C-terminus and utilizing the N-terminal region to screen theinhibitory sequence through a cloning approach. The inhibitory sequencesmay encompass both scFvs from the mAbs generated, as well as randompeptides and VHHs with randomized CDR3 regions. The luminogenicsubstrates may be further optimized for better resistance to non-BoNTproteases. The luminogenic substrates may incorporate sufficientsequences of SNAP25 to allow cleavage activation by BoNT/E, A and C. Theluminogenic substrates may also include a vesicle associated protein(VAMP) sequence to allow cleavage activation by BoNT/B, C and F. Themethod may also employ the use of bead immobilization of a fusionprotein containing full length firefly luciferase (FFL) protein and aBoNT specific cleavage site on nickel nitrilotriacetic acid (Ni-NTA)beads or Cyanogen bromide-activated Sepharose beads. The fusion proteinmay be immobilized to the beads via a lysine-rich anchor sequence. Whenthe fusion protein is bound to the beads, the FFL is substantiallyquenched when compared to the fusion protein in solution. Uponinteracting with BoNT, the proteolytic activity releases the FFL fromthe beads and increases the luminescence in the supernatant. This novelmethod may be used to detect proteolytic activity of additionalproteases when used with a protease specific cleavage site. For example,a cleavage site that can be recognized by the Tobacco Etch Virus (TEV)protease may be used as an optional control. Additionally, glycineand/or serine rich linker sequences may be added to both sides of theSNAP-25 sequence to enhance cleavage by BoNT. Luminogenic substrates forall toxins and enzymes as well as the seven serotypes (A to G) of BoNTmay be detected by such specific substrates. In certain embodimentstesting for different BoNT serotypes and subtypes in sterile filtratesof clinical isolates of Clostridium botulinium may be performed withluminogenic and fluorogenic substrates.

The novel substrates may also be obtained by the usual methods ofsolid-phase synthesis according to the Merrifield method on an automaticsynthesizer such as, for example, the 431A synthesizer from AppliedBiosystems. The chemistry used corresponds to Fmoc technology andprotection of the side chains allowing cleavage thereof withtrifluoroacetic acid, as described by E. Atherton and R. C. Sheppard(1989) in “Solid Phase Peptide Synthesis: a practical approach, IRLPress, Oxford.”

The ALISSA technology may also be applicable for use with targets suchas enzymes or toxins other than BoNT. For example, anthrax lethal factor(LF) has successfully been adapted for use in the ALISSA. As previouslydescribed, anthrax LF is a component of anthrax toxin that exhibits zincmetalloproteinase activity. The ALISSA is capable of quantitativelydetecting anthrax LF and reaching femtomolar sensitivities. Thus, ALISSAhas the potential to significantly improve the diagnosis of botulism,anthrax infection and potentially other serious infections, and couldserve to protect humans in biomedical and biodefense scenarios.Additionally, a specific and sensitive assay for the detection of LF ispotentially useful for early diagnosis of anthrax infection and isexpected to be a useful research tool to advance the understanding ofthe mechanism of action of anthrax toxin. Additionally, the ALISSAtechnology may also be extended to detect human chitinases (e.g. CHIT1and AMCase) and non-metalloproteases (Pep1 and Pep2 of Aspergillusfumigatus).

According to some embodiments, ALISSA may be used to detect systemicBoNT, such as toxin distribution in animal sera and organs. For example,BoNT levels may be measured in serum and organs of BoNT intoxicatedmice. In some embodiments, BoNT ALISSAs may be used to measure thesystemic content and tissue distribution in sub-lethallyBoNT-intoxicated mice. In certain embodiments the pharmacokinetic andserotype-dependent toxin distributions may be determined in organsfollowing parenteral or oral intoxication.

In certain embodiments, the pharmacokinetics of BoNT/A intoxication maybe studied using ALISSA. For example, the pharmacokinetic measurementson BoNT intoxication in the mouse model may be used to study thepersistence of sub-lethal concentrations of BoNT over extended periodsof time. Serum samples from intravenous (i.v.) and intragastric (i.g.)intoxication mice models may be used to study the toxin concentrationsduring a time course study. This method may be useful due to thewidening medical use of BoNT against conditions such as dystonia,depressions, and migraine, and the difficulties associated with such use(Crowner 2007; Jankovic 2004). Furthermore, luminogenic substrates mayalso be used to detect BoNT during a time course experiment in miceintoxicated with BoNT.

In some embodiments, the BoNT in intoxicated neurons may be quantifiedusing ALISSA. For example, ALISSA may be used to studyBoNT/A1-intoxicated rat hippocampal primary neuronal cells. In otherexamples, ALISSA may be conducted with Neuro2A and M17 cells in vitro aswell as with isolated neurons.

According to some embodiments, ALISSA may also be useful for detectionof botulism in clinical specimens. For example, ALISSA may be used todetect BoNT in sera from adult patients that have food-borne or woundbotulism. In other examples, ALISSA may be used to detect BoNT in serafrom infant patients with botulism.

EXAMPLES Example 1 Materials and Methods

The pure 150 kDa BoNT A toxin (holotoxin) was purchased from twodistinct commercial sources: from the List of Biological Laboratories(Campbell, Calif.) and Metabiologics Inc. (Madison, Wis.). The BoNT/Acomplex, IP and IV mouse assays in 50 mM citrate buffer, pH 5.5 wasreceived from Dr E. Jonson's laboratory, Food Research Institute of theUniversity of Wisconsin-Madison. The intact BoNT/A toxin complex andBoNT/A toxoid were from MetaBiologics. SNAPtide™ (FITC/DABCYL),synthetic peptide containing the native cleavage site for Botulinumtoxin type A and SNAPtide®, unquenched calibration peptide for SNAPtide™(FITC/DABCYL), were purchased from the List of Biological Laboratories.The latter contains the FITC bound to the N-terminal cleaved fragment ofSNAPtide; it was used as a calibrant to convert fluorescence intensityunits to changes in the molar ratio of peptide cleavage product. Alltypes of BoNT/A toxin were from Hall A, Clostridium botulinum producingstrain. In one example, the BoNT/A subtype used was A1. Toxin activitiesfor the holotoxin and the complex were 2.1×10⁸ MLD₅₀/mg and 3.6×10⁷MLD₅₀/mg, respectively, according to Metabiologics.

In certain embodiments, the fluorogenic peptide is SNAPtide (U.S. Pat.No. 6,504,006) which is a molecular derivative of SNAP25, the naturalsubstrate of BoNT/A. SNAPtide is cleaved by BoNT/A between a fluorophoreand a quencher (FRET pair) releasing unquenched fluorophore. TheSNAPtide contains a conjugated fluorescein thiocarbamoyl (FITC) quenchedby a 4-(dimethylaminoazo)benzene-4-carboxyl (DABCYL)-moiety. Thefluorogenic peptide SNAPtide (FITC/DABCYL, product #521) and theunquenched calibration peptide, containing an N-terminally FITC-labeledfragment of SNAPtide (product #528, synthetic, but sequence identical tothe BoNT/A cleaved product), were from List Biological Laboratories. Inother embodiments, the substrate comprises a SNAPtide peptide whereinthe N-terminal fluorescein isothiocyanate was replaced with 5-carboxyfluorescein. Such labeling improves stability of the substrate. Incertain embodiments, 4-methylumbelliferone labeling was utilizedallowing use of a substrate having blue fluorescence.

Affinity purified Rabbit polyclonal to Clostridium botulinum A Toxoid(formaldehyde inactivated Type A Neurotoxin (C. botulinum) antibodieswere purchased from Abcam (Cambridge, UK). Purified rabbit IgG was from(ICN Biomedical Inc., Aurora, Ohio), Seize® X Protein AImmunoprecipitation Kit was from Pierce (Rockford, Ill.), Trypsin wasfrom Promega, Fetal Bovine Serum was from Invitrogen (Carlsbad, Calif.).Human serum was from Sigma (cat. #H4522) and carrot juice was fromBolthouse Farms (Organics, 100% carrot juice, 1 liter bottle). Otherreagents were from Sigma unless indicated. Concentrations of the toxinswere determined according to the extinction coefficient (Ahmed et al.,2001) or by Bicinchoninic Acid (BCA, Pierce) Protein Assay, a MicroAssay for dilute protein solutions, with BSA as standard. Both methodsgave the same result. The product was exclusively the dichain form ofthe toxin as judged by the 12% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis at room temperature (25° C.) under reducing conditions.Gels were analyzed by Western Blot using Rabbit polyclonal toClostridium botulinum A Toxoid (Abcam). The bands on the gels werevisualized by Coomassie Blue or Silver staining or with the SimplyBlueSafeStain kit from Invitrogen (Carlsbad, Calif.).

Example 2 Assay Design

Preparation of the Immunomatrix.

BoNT/A was bound and enriched on a bead-based immuno-affinity matrix andcleansed from unspecific binding molecules. The enrichment matrixconsisted of protein-A conjugated sepharose beads coupled andcross-linked to polyclonal anti-BoNT/A antibodies (FIG. 1 a).Anti-BoNT/A antibodies were bound and then covalently cross-linked tothe bead-immobilized protein A molecules to prevent bleeding off whenmixed with sample. The release of antibodies into the sample mayotherwise lower the sensitivity of the assay. Capture antibodies weredirectly and covalently linked to the Protein A support (agarose beads)using the SeizeX Immunoprecipitation Kit (Pierce) as described in thesupplier's protocol. Briefly, 125 μg of Clostridium botulinum A Toxoid,formaldehyde inactivated Type A neurotoxin (ab 20641) prepared in 0.4 mLof Binding/Wash buffer contained 0.14 M NaCl, 0.008 M sodium phosphate,0.002 M potassium phosphate and 0.01 M KCl, pH 7.4, were bound andimmobilized to a Protein A support using cross-linker Disuccinimidylsuberate (DSS). 25 μL DSS, dissolved in DMSO immediately before use, wasadded to the spin cup containing the bound antibody support and gentlymixed for 30-60 minutes, centrifuged and washed 4 times with 500 μL ofImmunoPure Gentle Elution Buffer (Product No. 21027) with a high-salt,neutral pH elution system to quench the reaction and to remove excessDSS and uncoupled antibody. Alternatively, the beads were centrifugedand washed three times with 500 ul Gentle Elution Buffer and two timeswith 500 ul Gentle Binding Buffer (Pierce Product No. 21030). TheHandee™ Spin Cup Columns with functionalized beads were wrapped withlaboratory film to prevent gel from drying and stored at 4° C. untilused in experiments. The number of beads varying in sizes between 10-100μm was estimated microscopically with Reichert Bright-Line MetallizedHemacytometer (Hausser Scientific, USA). The immobilized polyclonalrabbit antibody used herein exhibited binding affinity to the light andheavy chain of BoNT/A, as confirmed by Western blot (FIG. 2 b).

BoNT/A Assay with a Large Immuno-Sorbent Surface Area (ALISSA).

The enzymatic activity of the immobilized BoNT/A was determined bycleavage of the specific fluorogenic substrate SNAPtide, a synthetic,commercially available, 13-amino acid peptide that contains the nativeSNAP-25 cleavage site for BoNT/A (Schmidt 2003; U.S. Pat. No. 6,504,006)(FIG. 1 b). BoNT/A holotoxin or its complex, were pre-incubated (whereindicated) in 5 mM dithiothreitol (DTT) for 30 minutes at 37° C. inorder to reduce and pre-activate the enzyme, immediately followingreconstitution in the buffer containing 20 mM HEPES, pH 8.0, 0.05%Tween-20, 0.3 mM ZnCl₂, and 1.0 mg/mL BSA to recover of BoNT/A.Pre-activated toxin was used immediately after reconstitution with thebuffer. Enzyme was immobilized on the antibodies-bound beads in 1 mL of3% Carnation non-fat milk in PBS or in a 10% Fetal Bovine Serum (heatinactivated). After the addition of immunomatrix samples were incubatedwith the food matrix or serum. Suspensions were rotated for indicatedtimes at 8 rpm on a Labquake Shaker/Rotisserie (Barnstead International,Dubuque, Iowa).

The antibody-bound beads with immobilized enzymes were separated by thecentrifugation, washed three times with 500 μl PBS buffer, collected andre-suspended in 300 μl of 20 mM HEPES buffer, containing 0.3 mM ZnCl₂,1.25 mM DTT and 0.1% TWEEN-20, pH 8.0. Unbound material was then removedby washing and synthetic peptide was added. Reaction was started byrotating and adding various concentrations of SNAPtide. For assays 4 mMDMSO-stock solution was diluted in 20 mM HEPES, pH 8.0, and a 100 μMstock solution was used. Assays were performed for indicated times in adark; reaction was stopped by transfer to ice and addition of a 20 mMethylenediaminetetraacetate (EDTA) (pH 8.0), which was equally effectivein blocking the proteolytic activity of both holotoxin and complexedBoNT/A. For work without antibodies-bound beads, the toxin was combinedwith the same number of beads with no antibodies or with the rabbitIgG-beads.

Conversion of SNAPtide releases the N-terminal fluorophore,fluorescein-thiocarbomoyl (FITC), which is initially quenched by theC-terminal chromophore, 4-((4-(dimethylamino)phenyl)azo)benzoic acid,succinimidyl ester DABCYL, was recorded with Wallac 1420 MultilabelCounter Victor²™ spectrophotometer (Perkin Elmer) or with SpectraMax M2(Molecular Device Corp.) at 485 nm/535 nm as an excitation and emissionwavelengths, respectively. The increase in fluorescence intensity isdirectly proportional to the amount of cleavage that has occurred andthus allows for accurate measurement of botulinum toxin enzymaticactivity. Before experiments the beads with cleaved SNAPTide wereobserved on the glass slides mounted on fluorescent microscope (OlympusBH2-RFCA, Japan). The immobilized polyclonal rabbit antibody used hereindid not significantly inhibit the specific proteolytic activity ofBoNT/A (FIG. 2 a).

Example 3 Assay Optimization

The conditions of bead-based assay were optimized to maximize itssensitivity and specificity for one- and ten-attomolar toxinconcentrations in 1-mL sample volumes (see FIGS. 3 a-e)). Therefore,assay sensitivity was repeatedly measured using serial dilutions ofBoNT/A in 10% fetal bovine serum (FBS), and the following parameterswere evaluated: antibody-protein A cross-linking conditions, postcross-linking bead wash buffers, number of beads, toxin-antibody bindingtimes and temperature, wash buffers for the removal of non-specificantibody binders, SNAPtide concentration, SNAPtide conversion time andthe effect of temperature during reaction. For each BoNT/A dilution, thefluorescence intensity was plotted against the variant parameter. Due tothe asymptotic nature of the resulting curves, there are no optima forseveral parameters. However, by analyzing the change in signal gain as afunction of a given parameter, efficient values for each parameter whichproduce the steepest increase in signal gain have readily been achieved,and the assay's performance has become robust and predictable.

During synthesis of the bead-based immunomatrix, use of a neutral washbuffer after binding and cross-linking of the antibody to the protein Abeads was found to be critical. When an alternative acidic wash buffer(pH 2.8) was used instead, the antibodies were altered such that theybecame inactive when exposed to nanomolar concentrations of the toxin,probably through toxin-induced proteolytic cleavage.

The assay performed efficiently and with high sensitivity when usingabout 100 μL/well SNAPtide in concentrations of about 25 μg/mL (FIG. 3a) and 1 uM. A reaction time of one hour for the conversion of thefluorogenic SNAPtide at room temperature (25° C.) was appropriate (FIG.3 b). Within limits, the signal-gain of the assay can be enhanced byincreasing the number of beads mixed with the sample (FIG. 3 c) and byextending the enzyme enrichment time as demonstrated in FIG. 3 d. Themost efficient bead concentrations lie between about 100,000 and about120,000 beads/mL, which correspond to a bead bed volume of approximately8.7-10.4 μl or approximately 10-12 μl, when left to settle. Furtherincrease of the bead concentration to 500,000/mL raised the signalintensity only by another 28% (FIG. 3 c). The effect of temperature onthe toxin capturing step was also investigated. Sufficient bindingrequired about 3 hrs at about 25° C. incubation for the substrate withthe beads, and about one hour when at about 37° C. The measured toxinactivity was significantly diminished at about 55° C. (FIG. 3 e), likelydue to toxin deactivation rather than due to decreased antibody binding.An increase in temperature from about 25° C. to about 37° C. during theSNAPtide-conversion reaction improved the signal and reliable readingswere obtained for reaction times of about 1 hour.

Pre-activation of the toxin on the beads during the wash step wasachieved with 5 mM DTT and produced slightly higher signals whencompared to the non-pre-activated toxin (FIG. 4A). However, thesubsequent reaction with the fluorogenic reporter had to be performed in1.25 mM DTT in order to avoid denaturation of the immunoaffinity matrixand because prolonged exposure to the more concentrated reductive agentinactivated the toxin considerably (tested on bead-free toxin).

Stable BoNT-Immobilization Matrix.

Previously, the antibody-coated beads could not be stored for more thantwo weeks in liquid suspension without experiencing a significant lossof ALISSA sensitivity. Suitable conditions for the extended dry storageof lyophilized ALISSA beads were determine. When freshly reconstitutedin liquid buffers, no substantial loss of sensitivity was observed after3 month of bead storage. Key to the longevity of the bead matrix was thechoice of suitable lyophilization conditions. All antibodies wereprovided by our collaborator, Dr. James D. Marks at the University ofCalifornia, San Francisco (UCSF).

Suitable Antibodies for BoNT ALISSAs.

Although originally developed with rabbit polyclonal antibodies thatrecognize both BoNT light chain (LC) and heavy chain (HC), monoclonalantibodies (mAbs) that bind certain BoNT epitopes are also usable in theALISSA. mAbs enhance specificity, and can positively affect BoNT'smetalloprotease activity. The functional and biochemical aspects ofantibodies used to develop the ALISSA technology into a reliable productwere investigated. More than 37 different monoclonal and polyclonalantibodies have been characterized for their suitability to be used inthe ALISSA. The BoNT-binding specificity was investigated and the effectthat antibodies exert on the enzymatic activity of antibody-bound BoNTin solution and after surface immobilization was measured. AlongsidemAbs provided by the PSWRCE members Dr. Larry Stanker (USDA) and Dr.James Marks (UCSF), a number of murine mAbs that were generated againstBoNT LC/A peptides, recombinant (r) LC/A and B, and BoNT toxoid(formaldehyde inactivated complex) were evaluated.

Although both anti-LC and anti-HC mAbs function in the ALISSA, anti-LCantibodies are preferred for most sample matrices that require stringentwash steps. Interestingly, some mAbs enhance the catalytic activity ofLC in a bead-free reaction, while others have no catalytic influence orare inhibitory. Certain mAbs bind LC with high affinity, but do notrecognize the LC that is present in the BoNT complex. The mAbs that aremost suitable for the ALISSA bind LC both when incorporated in the BoNTcomplex and when free in solution. One polyclonal (old Abcam batch ofab20641) and three mAbs were functional in the BoNT/A ALISSA. One mAbfrom Larry Stanker at the USDA was usable for the BoNT/B ALISSA, and onepolyclonal antibody from Metabiologics provided acceptable results inthe BoNT/E ALISSA.

Antibodies reactive to LC or heavy chain (HC) of BoNT/A can be used inthe BoNT/A ALISSA (FIGS. 5A and 5B)). The monoclonal mouse antibodiesF1-5 against HC, and F1-40 against LC tested, were produced and providedby Dr. Larry Stanker at the USDA. Antibodies that inhibit the catalyticfunction of BoNT are not suitable for the ALISSA. For example the CDCequine polyclonal antibody used for serotyping did not enhance thesensitivity of the bead-based ALISSA (FIG. 6 A) and inhibited BoNT/A'scatalytic activity in solution (FIG. 6 B).

In some embodiments, polyclonal, monoclonal antibodies and immobilizedsingle chain Fragment variables (scFvs), derived from mAbs, may beselected such that they bind BoNT light chains (LCs) with nanomolar (orbetter) affinity and without inhibiting the catalytic activity of BoNT.In certain embodiments, the antibodies bind BoNT LC epitopes in BoNTcomplex and holotoxin with similar affinity. In other embodimentssurface plasmon resonance may be used to measure binding affinities aswell as ALISSA assays with immobilized affinity reagents to determinedetection limits and linearity for BoNT detection. The pharmacokineticsof these affinity reagents may be tested for the analysis of BoNT fromdifferent clinical and medically relevant samples. In certainembodiments the BoNT/A affinity reagents may be used in a research-gradeproduct prototype to be used for the regulatory approval process.

Comparison of Agarose and Magnetic Beads.

A comparison between Protein A coated agarose and magnetic beads wasperformed to determine the sensitivity of each bead type. Agarose beads(6%) are approximately 10-100 μm in diameter (exclusion limit forproteins ˜5,000 kDa); whereas, magnetic Dynabeads, coated with ProteinA, are 2.8 μm in diameter and have a smooth surface. ALISSA resultsdemonstrated that the smaller magnetic Dynabeads were more sensitive athigher concentrations of BoNT/A complex (10⁻⁹ mol/L); whereas, thelarger agarose beads were more sensitive at lower concentrations ofBoNT/A complex (10⁻¹⁷ mol/L) (FIG. 7).

Optimization of ALISSA Immunomatrix Using a Double Affinity Matrix.

The ALISSA method not only immobilizes and thereby concentrates thecatalytic subunit of BoNT on the beads, it also substantiallyaccelerates the enzymatic turnover rate of the BoNT-substrate conversionby approximately 18-fold (bead based ALISSA: typical K_(m)=0.2 μM,V_(max)=14.5 μM/min/μg; non-bead based assay: typical K_(m)=0.7 μM,V_(max)=0.8 μM/min/μg). This enhancement of catalysis is accompanied bya weak interaction between substrate molecules and the bead surface. Tofurther improve enzyme acceleration by strengthening the substrate-beadinteraction, the conditions of the ALISSA immunomatrix were optimized toinclude a double affinity immobilization matrix.

An immobilization matrix was prepared that contained antibodiestargeting both BoNT/A (F1-40: monoclonal anti-BoNT/A light chain (LC)antibody) and the BoNT/A cleavable substrate containing an N-terminal5-carboxyfluorescein group (anti-FITC antibody). Remarkably, when bothanti-BoNT and anti-fluorescein antibodies were bound to beads, theALISSA signal was about two to four-times stronger than when anti-BoNTbeads alone were used (FIG. 8A and B, compare blue bars to orange bars,respectively). The effect was clearly visible throughout a dilutionseries of BoNT/A in serum (FIG. 8A and B). However, the effect was notobserved when the beads were coated only with the anti-fluoresceinantibody (FIG. 8A and B, green bars). Also, no further enhancement wasobserved when anti-BoNT and anti-fluorescein antibodies were mounted onseparate beads and then mixed before conducting the ALISSA experiment.

Higher fluorescence signals were achieved with the double affinitymatrix compared to the single affinity matrices (only anti-BoNT/A LC oranti-FITC) upon overnight (FIG. 8A) and 48 hour incubation (FIG. 8B)(except for the highest BoNT/A concentration point (10 ⁻⁹ mol/L) wherethe signal was comparable between the F1-40/anti-FITC combination andF1-40 alone for both time points). The results were similar when theexperiment was performed with supernatant alone (no beads) (FIG. 8C).These results demonstrate that the proximity of a controlled andpossibly strong substrate-bead interaction to a strong enzyme-beadinteraction can be exploited to enhance the turnover rate of thereaction of an immobilized enzyme.

A titration was performed using increasing concentrations of anti-FITCantibody to determine the most effective ratio of anti-BoNT/A LC andanti-FITC to use to provide an optimal signal to noise ratio for thedouble affinity immobilization matrix. An improved signal to noise ratiowas observed with increasing concentrations of anti-FITC antibody (FIG.9).

Example 4 Assay Performance: Sensitivity

The 150-kDa BoNT/A holotoxin (from two different commercial sources) andthe 500-kDa BoNT/A complex were serially diluted and tested by BoNT/AALISSA in 10% fetal bovine serum (FBS) (FIG. 4A). Significant signals ofseveral thousand relative fluorescence units (RFU) were still observedfor concentrations of one attomol/L in 1-mL sample volumes. Signals fortoxin complex were always stronger than for identical molarconcentrations of holotoxin. The practical detection limit in thediluted serum was extrapolated to be ˜0.5 attomol/L, which correspondsto 250 attogram toxin complex in 1 mL sample (FIGS. 3E, 4A). Todetermine ALISSA use in complex samples, the assay's sensitivity for thetoxin complex was also determined in spiked undiluted human serum,carrot juice, reconstituted non-fat powdered milk, fresh milk, andgelatin phosphate (GP) diluent (FIG. 4B). GP diluent is typically usedin the life mouse toxicity bioassay. Although somewhat lower than intoxin-spiked samples with 10% FBS, discernable fluorescent intensitiesabove background were still detected for 1 attomol/L toxin complex, withsignal intensities of ˜14,800, ˜14,750, ˜3100, ˜2500 and ˜650 RFU abovebackground in undiluted human serum, 50% carrot juice, GP diluent,non-fat milk and fresh milk, respectively. A fat-solubilizing washbuffer (with HEPES) was required for analyses of fresh milk samples.Overall, the ALISSA signals correlate proportionally with the toxinconcentration over several orders magnitude (FIG. 4A-B).

The ALISSA performed with comparable sensitivities in undiluted humanserum, 50% carrot juice (adjusted to pH 7.5 with binding buffer),reconstituted powdered milk, fresh milk and GP-diluent. In directcomparison with the mouse assay, the ALISSA was considerably faster and4-5 orders of magnitude more sensitive.

Example 5 Assay Performance: Specificity and Kinetics

Specificity of the assay and sensitivity and kinetics of the bead-basedALISSA compared to those of the bead-free conversion of the reporterpeptide were tested. To test non-specific agents, serum samples wereutilized with: 1) beads conjugated to purified nonspecific rabbit IgG;2) trypsin, because it is also able to cleave SNAPtide, but cannot beenriched on the beads; 3) BoNT type B complex; 4) BoNT type E complex;5) type A toxoid, which is a non-toxic, antibody-binding formaldehydeinactivated derivative of BoNT/A; and 6) a toxin-free control (FIG. 10a, 10b). The bead-based assay produced low intensity signals with thenon-specific agents trypsin, BoNT/A toxoid, BoNT/B and E) and only atthe highest tested concentrations of 10-100 pmol/L. The bead-freereaction mixture yielded signals only with trypsin and BoNT/A forconcentrations of 1 pmol/L or greater, and these signals were weak.Equimolar trypsin concentrations led to even higher signals than theBoNT/A complex. Toxin type B and E complexes, for which the peptidesubstrate does not contain specific cleavage sites, produced very weaksignals only at the 10 and 100-picomolar concentrations that were evenlower than those obtained with the bead-based assay. Interestingly, thebead-based detection of BoNT/A produced significantly higher signals—atany given toxin dilution step—than did the bead-free reaction mixture.For the bead-free reaction mixture discernable signals were onlyobtained for BoNT/A concentrations greater or equal to 1 pmol/L. Incontrast, strong signals were obtained with BoNT/A at concentrations aslow as 1 attomol/L when used in the bead-based assay. For the comparisonof bead-free versus bead-based assay, toxin concentrations and totaltoxin amounts were identical in each dilution step.

This remarkable enhancement of the substrate cleavage reaction as aresponse to BoNT/A immobilization prompted a determination of thekinetic parameters of the SNAPtide conversion reaction. For comparativepurposes, fixed total BoNT/A complex concentrations of 100 pmol/L wereused in 1-mL sample volumes for both the reactions with the free andwith the bead-immobilized toxin. At this concentration, BoNT/A is safelydetected with either method. Kinetic constants were obtained from plotsof initial rates versus eight concentrations of substrate ranging 0.0125to 5 μM. (Blanch and Clark, 1997; Liu et al., 1999). Initial velocityfor reactions was calculated from linear regression analysis as μM ofcleaved SNAPTide/min/mg enzyme. The values are the averages of 4independent determinations±error propagation. The Km value for theBoNT/A was calculated from the Lineweaver-Burk double reciprocal plot.No-enzyme reference was applied in establishing baseline RFU when thereis no enzyme activity. This control contained all components of theBoNT/A reaction mixture except the enzyme, replaced with the equivalentvolume of reaction buffer. Background fluorescence was determined byusing wells with only SNAPtide. Results are the averages of triplicatedeterminations

The hydrolysis of SNAPtide by BoNT/A obeys Michaelis-Menten kinetics andis characterized by a linear relationship between the reciprocalsubstrate concentration and the activity of the enzyme (FIGS. 10 c, 10d). Michaelis constancies (K_(m)) and maximal conversion rates (V_(max))were calculated from the linear regression of the reciprocal SNAPtideconcentration 1/[SNAPtide] versus the reciprocal reaction rate (1/V).The K_(m) of the immobilized enzyme is 3.2-fold lower than for the freeenzyme, suggesting a slightly higher enzyme/substrate affinity.Interestingly, the main effect was found in the rate of catalysis: theimmobilized BoNT/A is capable of converting its substrate with an18-fold increased maximal conversion rate than the free toxin. Thecorresponding values for V_(max) at 25° C. were 0.79±0.04 μM/min/μg and14.49±0.27 μM/min/μg for free (non-immobilized) and immobilized enzymes,respectively.

Example 6 Assay Performance

Comparison of BoNT/A ALISSA with the Live Mouse Assay.

A split aliquot of 100 ng BoNT/A toxin complex was shipped in arefrigerated hazmat container to collaborators at the Infant BotulismTreatment and Prevention Program of the California Department of PublicHealth (CDPH) in Richmond for use in the diagnostic life mouse bioassay.Identical dilution series of the toxin in GP-diluent were preparedconcurrently in pre-prepared and weighed vials at both institutions. Theapproximate time of the i.p. mouse injections at the two locationscoincided by a margin of minutes. Mice weighing 18-22 g each wereinjected i.p. with 0.5 mL/mouse of sample and watched for signs ofbotulism or death for the standard 96 hour observation period. Theresults of BoNT/A ALISSA became available after ˜2.5 hours and mice wereobserved for three days (Table 2, FIG. 11).

The mouse assay was positive for the highest test concentrations of 300and 60 pg/mL (0.5 mL injected per mouse). Mild symptoms of botulismdeveloped within 96 hours in three of five mice that received onehundreds of the theoretical LD₅₀ (0.3 pg). All other animals thatreceived 10⁻⁴ or 10⁻⁵ LD₅₀ remained completely disease free andasymptomatic. BoNT/A ALISSA produced clear signals throughout thedilution series. The lowest BoNT/ALISSA fluorescence signal at thelowest test concentration was 0.6 fg/mL (10⁻⁵ LD₅₀), which is still˜3,100 units above background levels.

TABLE 2 Comparison of the mouse bioassay with BoNT/A ALISSA [complex]Mouse bioassay ALISSA result (fg/mL) LD₅₀s^(a) result (RFU) 300,000.0 55/5 dead in <4 hrs 51,105 ± 95 60,000.0 1 5/5 dead in <21 hrs 48,009 ±464 600.0 10⁻² 3/5 mild symptoms^(b) 28,049 ± 1713 6.0 10⁻⁴ 5/5 diseasefree^(b) 13,954 ± 1324 0.6 10⁻⁵ 5/5 disease free^(b)  3,116 ± 15 0.0 0n.d.    0 ± 8 ^(a)calculated per injected 0.5 mL sample; one LD₅₀ = 30pg BoNT/A complex; ^(b)all mice alive after 69 hrs; n.d., not determined

The BoNT/A ALISSA avoids interference with other sample components byusing a highly BoNT/A-specific affinity matrix and by using aBoNT/A-specific substrate to exploit the natural proteolytic activity ofthe toxin. Both steps also amplify the signal by 1) localized enrichmentof the toxin; and 2) through enzymatic conversion of billions ofsubstrate molecules per toxin molecule. The capture matrix is designedto stably enrich the toxin, while retaining its enzymatic activity andby purifying the toxin from other non-specific proteases contained inthe sample. The beaded-protein A matrix binds to the antibodies via theFc regions, orienting the antigen binding domains away from the beadsurface and into the surrounding sample fluid (FIG. 12). This provideshigher accessibility to target toxin molecules.

The plateaus observed in the assay's response curves used to optimizesubstrate concentration and size and volume proportions of theimmunosorbent matrix represent saturation effects that indicate when thesubstrate concentration is no longer rate-limiting. Antibody bindingcapacity was about 50 ug antibody per one million beads which estimatesto an antibody dissociation constant kD at half maximum saturation to beapproximately 15 nM. Use of antibodies having higher binding affinitywill increase assay sensitivity. High affinity anti-BoNT antibodies havebeen used as antitoxins to neutralize systemic botulinum toxin inbotulism patients (Marks 2004; Garcia-Rodriguez 2007). This mode of“neutralization” however, should not be confused with inactivation ofthe toxin's enzymatic activity by steric hindrance of the catalytic siteresulting from antibody binding. Antibody-mediated “neutralization” oftoxin in vivo depends on formation of antibody-antigen complex andhepatic accumulation and clearance (Ravichandran 2006; Simpson 2001).

Use of a standard curve to measure concentration-dependent intensity offluorescence signal of un-quenched calibration peptide (FIG. 13) alloweddetermination of the molar conversion rate for the substrate molecules.A calculated substrate conversion rate of approximately two billionsubstrate molecules per one immobilized toxin molecule per hour wascalculated for the 10 attomolar toxin concentration. The reaction beinglimited by the rate at which the toxin becomes inactivated. Factors suchas chelation of the zinc atom by DTT, denaturation of the toxin by thereducing buffer, or proteolytic degradation of the toxin either throughautoproteolysis or by a contaminant protease may also contribute toinactivation of the toxin.

In certain embodiments, optimal temperature is 37° C. coincident withthe temperature at which the natural action of the toxin occurs and atwhich IgG antibody binding may be optimal. Higher temperatures mayinactivate the toxin. Preferably, the pH of the sample is approximatelyneutral (between about 6 and about 8). Assay sensitivity may also befurther increased by reducing background fluorescence of uncleavedsubstrate such as uncleaved SNAPtide.

In certain embodiments, a peptide conjugated FRET pair with a2,4-dinitrophenyl acceptor and a 4-methyl-7-dimethylamino-coumarin donormay be used as a substrate. These and other FRET pairs having betterspectral overlap can allow lower background fluorescence with goodkinetic properties.

In certain embodiments, an approximately 18-fold increase in maximumconversion rate v_(max) and a three-fold higher affinity to thesubstrate (three-fold lower k_(M)) for the immobilized toxin wasobserved as compared to free toxin in solution (FIGS. 10C and 10D). Theaverage bead surface area in the ALISSA assay is approximately 7.85 cm²per sample (based on a 50 μm average bead diameter) whereas theantigen-binding surface area in a conventional solid-phase orsolid-state ELISA with a 96-well flat-bottom microplate measures onlyabout 0.256 cm² per well. Thus, the available reaction surface area inthe ALISSA is about 30-fold greater than provided by prior art methods.Such immobilized toxin is also better protected from proteolysis andaggregation. Molecules of unstable BoNT/A light chain are sufficientlyseparated to diminish any autocatalytic degradation. Use of bead-basedassay also allows for more stringent wash procedures thereby diminishinginterference by other proteases. This was demonstrated for BoNT/A whencompared to equimolar concentrations of trypsin, BoNT/B and BoNT/E. Theincreased reaction surface area and control of diffusion through morefrequent substrate-enzyme interactions also contributes to the improvedenzymatic activity.

Example 7 Fluorogenic Substrates

Fluorogenic peptides were synthesized using standard Fmoc chemistrymethodology well known in the art. Commercial SNAPtide (List BiologicalLaboratories) contains a fluorescein isothiocyanate (FITC)-labelledN-terminus and a thiourea group that is unstable over time or when inthe presence of acids. This can result in undesired background signal.To avoid this, different SNAPtide-like peptides that were N-terminallylabelled with either 5-carboxyfluorescein (5-FAM)- or4-Methylumbelliferone (4-MU) were synthesized. Each fluorophore isconjugated at or near the N-terminus region of the peptide via a peptidebond, which enhances the stability of the substrate. Additionally, thepeptides contain a dark quencher, DABCYL, conjugated at or near theirC-terminus. Upon excitation, the DABCYL suppressed the fluorescenceemission of the 5-FAM and 4-MU labeled peptides when the peptides werenot cleaved and the fluorescent label and DABCYL remained close together(FIG. 14). However, when the peptide was cleaved by BoNT/A, thefluorescent label and DABCYL were separated and the fluorescent labelemitted light energy upon excitation. Each of the novel peptidescontains arginines having an L-isomeric form.

The effect of the substrate's C-terminal amino acid corresponding toM202 in the native human SNAP-25 sequence (SEQ ID NO: 11) was alsodetermined. Commercial SNAPtide replaces the M202 in the native sequencewith a Norleucine residue. This Norleucine residue provided efficientcleavage of the substrate as deletion or replacement with6-aminohexanoic acid greatly diminished the efficiency of theBoNT/A-mediated cleavage reaction (FIG. 15). The Norleucine used is anon-oxidizable surrogate for the methionine residue located at 202.Peptides that contain the Norleucine residue and showed efficientcleavage by BoNT/A are:

(SEQ ID NO: 21) #115: 5-FAM-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys[DABCYL]-Nle; and (SEQ ID NO: 22)#116: 4-MU-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]-Nle;wherein Nle is Norleucine (Table 3).

Peptides that do not contain a Norleucine or 6-aminohexanoic acid andare not cleaved by BoNT are:

TABLE 4 (SEQ ID NO: 20) #110: 5-FAM-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys[DABCYL]; and (SEQ ID NO: 23)#111: 4-MU-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys [DABCYL].

Peptides #112 and #113 that contain 6-aminohexanoic acid in place of theNorleucine residue located at 202 that are not cleaved by BoNT/A asshown in FIG. 14 are:

(SEQ ID NO: 19) #112: 5-FAM-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys[DABCYL]-Hex; and (SEQ ID NO: 5)#113: 4-MU-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]-Hex;wherein Hex is 6-aminohexanoic acid (Table 5).

By applying a manual docking approach supported by functional assays,several novel substrates were produced (Table 3 (SEQ ID NO: 21 and 22),Table 6 (SEQ ID NO: 12), Table 7 (SEQ ID NO: 13), and Table 8 (SEQ IDNO: 14)). The chemical structures (identified as “1”, “2” and “3”) ofeach exemplary substrate is depicted below its corresponding tablelisting of amino acid sequence. In the below exemplary embodiments, eachof the novel peptide substrates contains 12 amino acid residues.

Additional control peptides that contain 6-aminohexaonic acid thatcannot be cleaved by BoNT were produced (Table 4 (SEQ ID NO: 20 and 23),and Table 5 (SEQ ID NO: 19 and 5)). In addition, control peptides havingan RA to EL mutation (indicated in bold text) were produced (Table 9;SEQ ID NOS: 15, 17-18). The BoNT/A protease cannot efficiently cleavethe control peptides while other proteases are able to cleave thesepeptides, making them suitable control peptides for use in the ALISSA.The control peptides allow for compensation of the background signalresulting from non-BoNT/A protease activity or non-target proteaseactivity.

TABLE 3 Cleavable Peptides: #115: [5-Fam]TRIDEANQRATK[DABCYL]-Nle (SEQID NO: 21) #116: [4-Mu]TRIDEANQRATK[DABCYL]-Nle (SEQ ID NO: 22) Number 1Letter Code Amino acid name and modification  1 [5-Fam]T or Threoninewith a 5-carboxyfluorescein or 4- [4-Mu]T Methylumbelliferone conjugatedto its α-amino group (as illustrated by SEQ ID NOS: 21 and 22,respectively)  2 R Arginine  3 I Isoleucine  4 D Aspartic acid  5 EGlutamic acid  6 A Alanine  7 N Asparagine  8 Q Glutamine  9 R Arginine10 A Alanine 11 T Threonine 12 K[DABCYL] Lysine with DABCYL conjugatedto its ε-amino group 13 Nle Norleucine with an amide C-terminus #115(SEQ ID NO: 21)

#116 (SEQ ID NO: 22)

TABLE 4 Control Peptides: #110: [5-Fam]TRIDEANQRATK[DABCYL] (SEQ ID NO:20) #111: [4-Mu]TRIDEANQRATK[DABCYL] (SEQ ID NO: 23) Number 1 LetterCode Amino acid name and modification  1 [5-Fam]T or Threonine with a5-carboxyfluorescein or 4- [4-Mu]T Methylumbelliferone conjugated to itsα-amino group (as illustrated by SEQ ID NOS: 20 and 23, respectively)  2R Arginine  3 I Isoleucine  4 D Aspartic acid  5 E Glutamic acid  6 AAlanine  7 N Asparagine  8 Q Glutamine  9 R Arginine 10 A Alanine 11 TThreonine 12 K[DABCYL] Lysine with DABCYL conjugated to its ε-aminogroup #110 (SEQ ID NO: 20)

#111 (SEQ ID NO: 23)

TABLE 5 Control Peptides: #112: [5-Fam]TRIDEANQRATK[DABCYL]-Hex (SEQ IDNO: 19) #113: [4-Mu]TRIDEANQRATK[DABCYL]-Hex (SEQ ID NO: 5) Number 1Letter Code Amino acid name and modification  1 [5-Fam]T or Threoninewith a 5-carboxyfluorescein or 4- [4-Mu]T Methylumbelliferone conjugatedto its α-amino group (as illustrated by SEQ ID NOS: 19 and 5,respectively)  2 R Arginine  3 I Isoleucine  4 D Aspartic acid  5 EGlutamic acid  6 A Alanine  7 N Asparagine  8 Q Glutamine  9 R Arginine10 A Alanine 11 T Threonine 12 K[DABCYL] Lysine with DABCYL conjugatedto its ε-amino group 13 Hex 6-Amino hexanoic acid #112 (SEQ ID NO: 19)

#113 (SEQ ID NO: 5)

TABLE 6 Cleavable Peptide: K[5-Fam]IDEANQRATK[DABCYL]Nle-amide (SEQ IDNO: 12) Number 1-letter code Amino acid name and modification  1K[5-Fam] Lysine with 5-carboxyfluorescein conjugated to its ε-aminogroup  2 I Isoleucine  3 D Aspartic acid  4 E Glutamic acid  5 A Alanine 6 N Asparagine  7 Q Glutamine  8 R Arginine  9 A Alanine 10 T Threonine11 K[DABCYL] Lysine with DABCYL conjugated to its ε-amino group 12 NleNorleucine with an amide C-terminus 1) K[5- Fam]IDEANQRATK[DABCYL.]-norleu-amide

TABLE 7 Cleavable Peptide Fam-K[5-Fam]IDEANQRATK[DABCYL]Nle-amide (SEQID NO: 13) Number Code Amino acid name and modification  15-Fam-K[5-Fam] Lysine with a 5-carboxyfluorescein conjugated to its αand ε-amino groups  2 I Isoleucine  3 D Aspartic acid  4 E Glutamic acid 5 A Alanine  6 N Asparagine  7 Q Glutamine  8 R Arginine  9 A Alanine10 T Threonine 11 K[DABCYL] Lysine with DABCYL conjugated to its ε-aminogroup 12 Nle Norleucine with an amide C-terminus 2)5-Fam-K[5-Fam]IDEANQRATK[DABCYL]-norleu-amide

TABLE 8 Cleavable Peptide:Alternative to above 2 substrates: 5-Fam-KIDEANQRATK[DABCYL]Nle-amide(SEQ ID NO: 14) Number Code Amino acid name and modification 1 5-Fam-KLysine with a 5-carboxyfluorescein conjugated to its α-amino group 2 IIsoleucine 3 D Aspartic acid 4 E Glutamic acid 5 A Alanine 6 NAsparagine 7 Q Glutamine 8 R Arginine 9 A Alanine 10 T Threonine 11K[DABCYL] Lysine with DABCYL conjugated to its ε-amino group 12 NleNorleucine with an amide C-terminus

TABLE 9 Control Peptides:5-Fam-K[5-Fam]IDEANQELTK[DABCYL]Nle-amide (SEQ ID NO: 15);5-Fam-KIDEANQELTK[DABCYL]Nle-amide (SEQ ID NO: 17); andK[5-Fam]IDEANQELTK[DABCYL]Nle-amide (SEQ ID NO: 18). Number CodeAmino acid name and modification 1 5-Fam-K[5-Fam];Lysine with a 5-carboxyfluorescein conjugated 5-Fam-K; orto either its α or ε-amino group or to both  K[5-Fam](there are three possibilities as illustratedby SEQ ID NOS: 15, 17 and 18) 2 I Isoleucine 3 D Aspartic acid 4 EGlutamic acid 5 A Alanine 6 N Asparagine 7 Q Glutamine 8 E Glutamic acid9 L Leucine 10 T Threonine 11 K[DABCYL]Lysine with DABCYL conjugated to its ε-amino group 12 NleNorleucine with an amide C-terminusThese exemplary control peptides cannot be efficiently cleaved bybotulinum neurotoxin serotype A, but can be cleaved by other proteases.Hence they can be used in the ALISSA assay as a control for non-specific(non-BoNT/A) protease activity. Below is the structure of one (SEQ IDNO: 18) of the three possible versions of the control peptides found inTable 9.

3) Control Peptide

By employing the above-described methods, several new substrates wereidentified for use in the ALISSA assay. The substrates exhibited higherchemical stability and high sensitivity for BoNT detection when used assubstrate. These substrates include, but are not limited to:

(SEQ ID NO: 12) Lys[5-Fam]IleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]Nle;(SEQ ID NO: 13) 5-Fam-Lys[5-Fam]IleAspGluAlaAsnGlnArgAlaThrLys[DABCYL]Nle; (SEQ ID NO: 14)5-Fam-LysIleAspGluAlaAsnGlnArgAlaThrLys[DABCYL]Nle; (SEQ ID NO: 21)(5-Fam)-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]Nle;(SEQ ID NO: 22) (4-Mu)-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]Nle;(SEQ ID NO: 16) LysIleAspGluAlaAsnGlnArgAlaThrLysNle; and(SEQ ID NO: 27) #203: Lys-(4-Mu)-IleAspGluAlaAsnGlnArgAlaThrLys[DABCYL]-Nle,

wherein Nle is Norleucine.

Peptides useful as control peptides were also generated by employing theabove-described methods. Said control peptides include, but are notlimited to:

(SEQ ID NO: 5) (4-Mu)-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]Hex;(SEQ ID NO: 15) 5-Fam-Lys[5-Fam]IleAspGluAlaAsnGlnGluLeuThrLys[DABCYL]Nle; (SEQ ID NO: 17) 5-Fam-LysIleAspGluAlaAsnGlnGluLeuThrLys[DABCYL]Nle; (SEQ ID NO: 18) Lys[5-Fam]IleAspGluAlaAsnGlnGluLeuThrLys[DABCYL]Nle; (SEQ ID NO: 19)(5-Fam)-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]Hex;(SEQ ID NO: 28) #204: Lys[5-Fam]IleAspGluAlaAsnGlnArgAlaThrLys[DABCYL]Hex; (SEQ ID NO: 29)#205: Lys[4-MU]IleAspGluAlaAsnGlnArgAlaThrLys [DABCYL]Hex;(SEQ ID NO: 20) (5-Fam)-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys  [DABCYL];and (SEQ ID NO: 23) (4-Mu)-ThrArgIleAspGluAlaAsnGlnArgAlaThrLys[DABCYL],

wherein Hex is 6-aminohexanoic acid and Nle is Norleucine.

The following table summarizes the BoNT/A cleavable and controlsubstrates provided herein:

TABLE 10 SEQ BoNT/A ID Peptide Cleavable  NO: # Code or Control 5 #113[4MU]-TRIDEANQRATK[DABCYL]-Hex-CONH₂ control 12 #201K[5-Fam]IDEANQRATK[DABCYL]-Nle-CONH₂ cleavable 13Fam-K[5-Fam]IDEANQRATK[DABCYL]-Nle-CONH₂ cleavable 145-Fam-KIDEANQRATK[DABCYL]-Nle-CONH₂ cleavable 155-Fam-K[5-Fam]IDEANQELTK[DABCYL]-Nle-CONH₂ control 175-Fam-KIDEANQELTK[DABCYL]-Nle-CONH₂ control 18K[5-Fam]IDEANQELTK[DABCYL]-Nle-CONH₂ control 19 #112[5FAM]-TRIDEANQRATK[DABCYL]-Hex-CONH₂ control 20 #110[5FAM]-TRIDEANQRATK[DABCYL]-CONH₂ control 21 #115[5FAM]-TRIDEANQRATK[DABCYL]-Nle-CONH₂ cleavable 22 #116[4MU]-TRIDEANQRATK[DABCYL]-Nle-CONH₂ cleavable 23 #111[4MU]-TRIDEANQRATK[DABCYL]-CONH₂ control 27 #203K[4MU]-IDEANQRATK[DABCYL]-Nle-CONH₂ cleavable 28 #204K[5FAM]-IDEANQRATK[DABCYL]-Hex-CONH₂ control 29 #205K[4MU]-IDEANQRATK[DABCYL]-Hex-CONH₂ control CON = amide C-terminus; 5FAM= 5-carboxyfluorescein; 4MU = 4-methylumbelliferone; Hex =6-aminohexanoic acid; Nle = Norleucine.

Example 8 Characterization of Fluorogenic BoNT Cleavable Substrates andControl Peptides for BoNT ALISSA

Specificity of Novel BoNT Cleavable and Control Peptides.

Other proteases have the ability to cleave the BoNT cleavable peptides;therefore, control peptides were produced for BoNT/A (see Example 7,peptides #110, 111, 112, and 113 (SEQ ID NOS: 20, 23, 19, and 5,respectively). The sequences of the BoNT cleavable peptide and thenon-cleavable control peptide are almost identical. They contain thesame amino acid residues (Q and R) at the BoNT/A cleavage site, butdiffer in their last residue, five amino acids from the cleavage site(norleucine replaced with its isomer, aminohexanoic acid). These controlsubstrates cannot be cleaved by BoNTs, but can be cleaved by somenon-BoNT-related proteases. Therefore, the control peptides were used inthe ALISSA assay as a control for non-specific (BoNT/A) proteaseactivity.

Cleavage specificity of the novel BoNT/A cleavable peptides (#115 (SEQID NO: 21) and #116 (SEQ ID NO: 22)) and control peptides (#110 (SEQ IDNO: 20); #111 (SEQ ID NO: 23); #112 (SEQ ID NO: 19); and #113 (SEQ IDNO: 5)) was tested using trypsin, BoNT/A complex, and BoNT/A and Bserotypes (FIG. 16). The control peptides were only cleaved by trypsinand not by BoNT/A complex (FIG. 16, left and middle panels). Moreover,the control peptide, #112, was not cleaved by addition of BoNT/A orBoNT/B serotypes (FIG. 16, right panel). This demonstrates that,together with the BoNT/A cleavable peptide substrates, the controlpeptides can be used to determine the specificity of the BoNT/A ALISSAand the extent of non-specific proteases in a sample.

Additionally, the BoNT/A cleavable peptide, #115, showed highspecificity for BoNT/A as demonstrated in the ALISSA using two differentBoNT serotypes. Peptide #115 was only cleaved upon addition of BoNT/Aserotype and not BoNT/B serotype (FIG. 16, right panel). This shows theremarkable specificity that BoNT/A cleavable peptides have for only theBoNT/A serotype.

Kinetic Analysis of BoNT Cleavable and Control Peptides.

An investigation of the kinetic properties of BoNT cleavable peptidesand control peptides was performed (FIGS. 17-18). As the nature ofprotease contaminants in a clinical sample is unknown, representativeproteases from the families of residue specific serine (trypsin),broad-spectrum serine (proteinase K), and zinc metallo-(thermolysin)proteinases were tested (FIG. 17). BoNT cleavable and control peptidesobeyed Michaelis-Menten kinetics when tested with all of the differentenzymes tested. Table 11 lists the kinetic affinity (Michaelis-Mentenconstant K_(M)), the catalytic turnover rates (k_(cat)), and thecatalytic efficiency (k_(cat)/K_(M)) for both BoNT-cleavable substratesand controls. This comparison of kinetic properties of the peptides inpresence of different proteases demonstrates that potential proteasesample contaminants cleave the controls with similar efficiency as theycleave the BoNT/A substrates.

TABLE 11 Enzyme/ BoNT/A- Control BoNT/A- Kinetic cleavable #112cleavable Control Parameters #115 (5-FAM) (5-FAM) #116 (4-MU) #113(4-MU) BoNT/A k_(cat), s⁻¹ 47.85 ± 2.44  — 49.39 ± 1.52 — K_(M), μM 4.99± 0.88 —  2.62 ± 0.37 — k_(cat)/K_(M), 9.59 ± 1.89 — 18.85 ± 3.26 — μM⁻¹s⁻¹ Trypsin k_(cat), s⁻¹ 98.98 ± 9.75  58.72 ± 4.17 42.78 ± 1.21 83.90 ±8.35 K_(M), μM 9.67 ± 2.15  3.75 ± 0.79  3.40 ± 0.29 13.26 ± 2.70k_(cat)/K_(M), 10.23 ± 1.52  15.66 ± 2.14 12.58 ± 1.14  6.33 ± 2.31 μM⁻¹s⁻¹ Thermolysin k_(cat), s⁻¹ 45.32 ± 3.52  24.26 ± 1.72 18.93 ± 0.6214.66 ± 0.71 K_(M), μM 8.16 ± 1.51  3.58 ± 0.76 12.66 ± 0.86  6.45 ±0.80 k_(cat)/K_(M), 5.56 ± 0.26  6.78 ± 1.89  1.5 ± 0.32  2.27 ± 0.79μM⁻¹ s⁻¹ Proteinase K k_(cat), s⁻¹ 45.89 ± 4.68  50.73 ± 3.28 18.11 ±1.11 30.64 ± 2.17 K_(M), μM 5.46 ± 1.49  3.37 ± 0.67  4.48 ± 0.78  4.87± 0.96 k_(cat)/K_(M), 8.40 ± 0.64 15.05 ± 2.89  4.04 ± 1.11  6.29 ± 2.05μM⁻¹ s⁻¹

A linear relationship between the signal responses of 4-MU and 5-FAMpeptides was observed in the presence of different proteases (FIG. 18).

Cleavability of Substrates and Controls with BoNT Subtypes A1 to A5.

As the BoNT ALISSA was originally developed for BoNT/A subtype A1, thepeptide substrates were tested to determine whether they could becleaved by other BoNT/A subtypes (A2 to A5). The BoNT/A peptidesubstrates were cleaved by all known BoNT/A subtypes except for A4 (FIG.19, A and B). None of the control peptides (#112, #113) were cleaved byany of the BoNT/A subtypes (not shown). The experiments were performedwith recombinant light chains (LCs) of A2 to A5 kindly provided by Dr.Andreas Rummel, MHH Institute for Toxicology, Hannover, Germany. The A4subtype does not cleave the BoNT/A-cleavable substrates nor thecommercial FITC-containing commercial SNAPtide (#521) from ListBiological Laboratories (Campbell, Calif.) (FIG. 19, A and B). However,LC A4 hydrolyzes a SNAP25 protein, BoNT/A natural substrate (FIG. 19,C). The remarkable reduction in cleavage rate of commercial SNAPtide byLC A4 was previously reported by others (Henkel 2009). In our hands LCA4 showed only 1.12% of activity with SNAPtide (#521) compared to LC A1(FIG. 19, A). In certain embodiments the ALISSA can be used to testother BoNT serotypes as well.

Zinc Metalloprotease Activity of BoNT.

The BoNT ALISSA measures the specific zinc metalloprotease activity ofimmunoaffinity-enriched BoNT. The BoNT cleavable and control peptideswere further tested in the ALISSA with protein NG agarose beads andrabbit polyclonal to BoNT/A toxoid antibody in spiked pooled humanserum. Only the cleavable substrate (#115, white bar) was cleaved uponaddition of increasing concentrations of BoNT/A complex, while thecontrol substrate (#112, black bar) showed only little cleavage byBoNT/A (FIG. 19 D). The zinc chelator,N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), suppressedBoNT metalloprotease activity (#115/TPEN, striped bar). This confirmedthat the concentration-dependent increase in fluorescent signal of BoNTcleavable substrate #115 was due to zinc metalloprotease activity ofbead-immobilized BoNT. The negligible fluorescent signal generated bycleavage of control substrate #112 indicated the presence ofnon-specific protease activity in the assay.

Substrate Cleavage by BoNT Light Chains.

The BoNT holotoxin consists of a heavy chain (HC) and a light chain(LC), which perform different functions. The BoNT HC binds the receptorand delivers the LC to its target substrate. The BoNT LC is responsiblefor the catalytic activity of BoNT by cleaving its specific substrate.Separately, the BoNT HC and LC components are not toxic. Therefore,substrates were identified that can be cleaved by the BoNT/A holotoxin,complex or the non-toxic LCs alone ((SEQ ID NO: 21 and 22), (SEQ ID NO:12), (SEQ ID NO: 13), and (SEQ ID NO: 14)). Cleavage by the non-toxic LCis a useful feature for the development of toxin free positive controlsfor the implementation of the ALISSA assay, e.g. in a research orclinical laboratory. In certain embodiments, the ALISSA may be used withBoNT light chains of known subtypes for BoNT A and B.

This set of substrates represents an exemplary complete set of BoNTcleavable substrates and internal controls for an endopeptidase-basedBoNT detection assay. No other endopeptidase-based BoNT assay containsBoNT-specific controls and allows discriminating non-BoNT specificprotease activity from BoNT-specific activity. Therefore, this assaydesign represents an important step towards future clinical applicationof the BoNT ALISSA on clinical specimens.

In some embodiments the control peptides can be used in combination withthe BoNT peptide substrates in the same sample. For example, controlpeptide #112 (5-FAM) can be used in combination with BoNT/A cleavablesubstrate #116 (4-MU), and control peptide #113 (4-MU) can be used incombination with BoNT/A cleavable substrate #115 (5-FAM).

Example 9 Novel Fluorogenic Substrate for BoNT/B and BoNT/E

A novel fluorogenic substrate for a BoNT/B ALISSA was designed andsynthesized at the City of Hope core facility for peptide synthesis. TheBoNT/B substrate contains the BoNT/B cleavable sequence ofvesicle-associated membrane protein (VAMP) (Table 12, FIG. 20, SEQ IDNO: 24). This substrate was used in an ALISSA to test for BoNTspecificity of substrate cleavage using BoNT/A and BoNT/B recombinantlight chains (LCs). The BoNT/B LC demonstrated remarkable specificityfor the substrate compared with the BoNT/A LC that did not cleave thesubstrate (FIG. 21 A). Additionally, specificity of the BoNT/B substrateto BoNT/B complex was demonstrated in a bead-based ALISSA and a beadfree reaction (FIGS. 21 B and C, respectively). This substrate is auseful feature for the development of a toxin-free positive control forthe implementation of the BoNT/B ALISSA assay because it can be usedwith the non-toxic BoNT/B LC. In certain embodiments a control peptideis used in the ALISSA for BoNT/B.

TABLE 12 BoNT/B Cleavable SubstrateK[5-Fam]LSELDDRADALQAGASQFETSAAKLKRK[DABCYL]-amide (SEQ ID NO: 24)Number Code Amino acid name and modification  1 K[5-Fam] Lysine with a4-Methylumbelliferone conjugated to its ε-amino group  2 L Leucine  3 SSerine  4 E Glutamic acid  5 L Leucine  6 D Aspartic acid  7 D Asparticacid  8 R Arginine  9 A Alanine 10 D Aspartic acid 11 A Alanine 12 LLeucine 13 Q Glutamine 14 A Alanine 15 G Glycine 16 A Alanine 17 SSerine 18 Q Glutamine 19 F Phenylalanine 20 E Glutamic acid 21 TThreonine 22 S Serine 23 A Alanine 24 A Alanine 25 K Lysine 26 L Leucine27 K Lysine 28 R Arginine 29 K[DABCYL] Lysine with DABCYL conjugated toits ε-amino group SEQ ID NO: 24

The ALISSA has also been expanded to detect BoNT/E, spiked in pooledhuman serum, using a combination of a commercial substrate (SNAP Etide)and a suitable antibody. The BoNT/E ALISSA reaches femtomolarsensitivity (FIG. 22). The commercial BoNT/E substrate SNAP Etideincorporates ortho-Aminobenzoic acid (o-Abz) as a fluorescence donor and2,4-dinitrophenyl (Dnp) as an acceptor (quencher). o-Abz has a muchlower quantum yield than coumarin or fluorescein derivatives, which ismost likely limiting the sensitivity of the newly developed BoNT/EALISSA. In one embodiment the BoNT/E ALISSA may be improved by modifyingthe chemistry of the substrate's fluorophore/quencher pair. In certainembodiments a fluorogenic cleavable and control substrate may bedesigned and used in the ALISSA to detect all BoNT serotypes, A to G.

Example 10 Bioluminescent Substrates

A series of protein engineering experiments were conducted to determinewhether extended sequences of recombinant SNAP25 (FIG. 60) can beexpressed in bacteria and remain cleavable by BoNT. Experiments werealso conducted to determine whether recombinant firefly luciferase (FFL)with C-terminal histidine tags or other modifications remain functional.Functional recombinant SNAP25 (rSNAP25) was expressed using commercialbrain cDNA library. Gel-shift experiment demonstrated that rSNAP25 isreadily cleaved by BoNT/A (FIG. 23) while having a C-terminalhexahistidine tag. The rSNAP25 was also cleaved at a faster rate ascompared to fluorogenic peptide SNAPtide. To generate recombinant FFL, afirefly luciferase yeast plasmid (pGAL-FFL) was obtained from a genedepository and used as a template to construct a pET-vector based onrecombinant firefly luciferase expression system. The recombinant FFLhad a C-terminal hexahistidine tag and was readily expressed in E. coli.When FFL-expressing bacteria were combined with 5-fluoroluciferin, theresulting light signal was strong enough to be visible with the nakedeye indicating that the protein was functional. The signal exceeded themaximally permissible luminescence signal strength of a Victorluminometer plate reader.

Fluorogenic peptide library for producing substrates was establishedusing previously described methods (Aina 2007; Juskowiak 2004; Rosse2000). Synthetic peptide libraries are generated using natural andnon-natural or non-proteinogenic amino acids to improve resistancetoward non-target proteases. As described further below, a beadedsynthesis resin support was used to perform a one-bead-one-compoundapproach in which each bead contains only one type of peptide inpicomolar quantities. The method used was as described previously (Aina2005; Lam 2003). On-bead conversion of the substrate was performed forseveral cycles of selection by first incubating the peptide bead librarywithout BoNT in presence of a relevant sample type (e.g. serum orhomogenized mouse organs) for extended periods of time. Peptidescontaining unstable peptides become fluorescent and were removed byparticle sorting using a flow cytometer. The beads that remainednon-fluorescent were then exposed to BoNT. Beads that became rapidlyfluorescent in presence of BoNT were separated with either amicromanipulator (tetrad dissection microscope at COH) or flowcytometer. The library was constructed such that the peptides can bedissociated from the beads readily (e.g. by CNBr cleavage) in order toallow decoding of the sequence of the fluorogenic substrate by massspectrometry.

Example 11 One-Bead-One-Peptide Libraries

One-bead-one-peptide libraries were generated by Fmoc(fluoren-9-ylmethoxycarbonyl) chemistry. Using a split and mix approach,the synthetic peptides begin with a block of 3 D-amino acids followed by5 randomized L-amino acids and another block of 3-D-amino acids.Addition of 3 D-amino acids to the N- and C-termini of a peptideincreases its stability. Natural proteinogenic, D-amino acids, rare aswell as unnatural amino acids (Table 13) are used. The number of beadsused is about three times greater than the number of possiblepermutations to obtain statistically relevant and reproducible results.For example, if 13 different amino acids are used for synthesis ofrandomized 5-mer peptides, 1.6 billion compounds are obtained andtherefore 3.6 billion beads are screened. When testing a suitable linkerwith an existing substrate, where an undesired amount of the cleavedfluorophore containing peptide remains on the resin beads used togenerate the library, the linker will be tested with existing substrateand if desired, the order of fluorophore and quencher can be reversed.Decoding of the novel fluorogenic substrate sequence is then performedby mass spectrometry analysis.

Listed below in Table 13 are commercially available (e.g. SigmaChemicals), non-natural amino acids that are suitable for use ingenerating one or more library of fluorogenic peptides.

TABLE 13 Short name Full name, synonym (S)-Fmoc-4-chloro-β-(S)-3-(Fmoc-amino)-4-(4-chlorophenyl)butyric Homophe-OHacidFmoc-4-chloro-L-β-Homophe-OH (R)-Fmoc-4-fluoro-β-(R)-3-(Fmoc-amino)-4-(4-fluorophenyl)butyric Homophe-OH acid,Fmoc-4-fluoro-D-β-Homophe-OH (S)-Fmoc-3-cyano-β-(S)-3-(Fmoc-amino)-4-(3-cyanophenyl)butyric Homophe-OH acid,Fmoc-3-cyano-L-β-Homophe-OH (S)-Fmoc-3-methyl-β-(S)-3-(Fmoc-amino)-4-(3-methylphenyl)butyric Homophe-OH acid,Fmoc-3-methyl-L-β-Homophe-OH (S)-Fmoc-3- (S)-3-(Fmoc-amino)-4-[3-trifluoromethyl- (trifluoromethyl)phenyl]butyric acid, Fmoc-3-β-Homophe-OH (trifluoromethyl)-L-β-Homophe-OH (S)-Fmoc-2-(S)-3-(Fmoc-amino)-4-[2- trifluoromethyl-(trifluoromethyl)phenyl]butyric acid, Fmoc-2- β-Homophe-OH(trifluoromethyl)-L-β-Homophe-OH (S)-Fmoc-4,4-(S)-3-(Fmoc-amino)-4,4-diphenylbutyric acid, diphenyl-β-Fmoc-4,4-diphenyl-D-β-Homoala-OH Homoala-OH (S)-3-(Fmoc-Fmoc-4-(2-naphthyl)-L-β-Homoala-OH, Fmoc-β- amino)-4-(2- 2-Homonal-OHnaphthyl)butyric acid (R)-Fmoc-4-(3-(R)-3-(Fmoc-amino)-4-(3-pyridyl)butyric acid, pyridyl)-β-Fmoc-4-(3-pyridyl)-D-β-Homoala-OH Homoala-OH (S)-3-(Fmoc-amino)-5-Fmoc-5-phenyl-L-β-norvaline phenyl-pentanoic acid (R)-3-(Fmoc-amino)-Fmoc-5-phenyl-D-β-norvaline 5-phenyl-pentanoic acid(S)-3-(Fmoc-amino)-5- Fmoc-4-vinyl-L-β-Homoala-OH hexenoic acid(S)-3-(Fmoc-amino)-6- Fmoc-4-styryl-L-β-homoalanine phenyl-5-hexenoicacid (S)-Fmoc-3,4-difluoro-(S)-3-(Fmoc-amino)-4-(3,4-difluorophenyl)butyric β-Homophe-OH acid,Fmoc-3,4-difluoro-L-β-Homophe-OH (S)-Fmoc-4-chloro-β-(S)-3-(Fmoc-amino)-4-(4-chlorophenyl)butyric Homophe-OHacidFmoc-4-chloro-L-β-Homophe-OH Fmoc-4-Abz-OH 4-(Fmoc-amino)benzoicacid (R)-Fmoc-4-(3- (R)-3-(Fmoc-amino)-4-(3-pyridyl)butyric acid,pyridyl)-β- Fmoc-4-(3-pyridyl)-D-β-homoalanine Homoala-OH4-(Fmoc-aminomethyl) 4-(Fmoc-aminomethyl)benzoic acid benzoic acidFmoc-homocyclo- 1-(Fmoc-amino)cyclohexanecarboxylic acid leucineFmoc-β-(3-benzo- Fmoc-3-(3-benzothienyl)-D-alanine thienyl)-D-Ala-OH

Use of purified fusion proteins as substrates will allow for furthercharacterization of the novel fusion substrates. Mass spectrometry andgel electrophoresis will be used on the purified proteins to measure theefficiency of the cleavage reaction.

Example 12 Identification of Luminogenic Protein Substrates

Genetically engineered variants of recombinant luciferase protein thatbecome activated by specific cleavage reactions are obtained using thefollowing strategies: 1) complementation of inactive luciferasefragments to restore active luciferase molecules; and 2) specificreactions to release the D-luciferin as a substrate for fireflyluciferase (FFL from Photinus pyralis).

Split FFL constructs are designed and used to detect presence of aspecific proteolytic activity. Mutants of FFL can be used to emit lightat distinct wavelength varying from greenish-yellow (˜560 nm) to orangeand red (605 to 613 nm) which allows multiplex detection of severalagents on a single device. Also, the color of the light emitted byRenilla reniformis (sea pansy) luciferase (RLuc) can be tuned by thechemical environment in which the light-emitting coelenterazineoxidation is performed (Miyaki et al., “Bringing bioluminescence intothe picture,” Nature Methods, 4:616-617 (2007)).

Using this cloning strategy, a set of rSNAP-25/FFL fusion constructswere generated with pETblue-2 and pET28a expression vectors. Becauseco-expression of overlapping split FFL domains reconstitutes active FFL,an overlapping FFL fusion construct that is interrupted by an integralSNAP-25 sequence containing the BoNT/A cleavage site (within SNAP-25residues 187 to 206) was cloned and expressed. The resulting fusionprotein encompasses FFL[1-475]SNAP-25[187-206]FFL[265-550] (bracketsdenote the amino acid ranges). In addition an overlapping FFL fusionprotein encompassing FFL[1-478]SNAP-25[187-206]FFL[265-550] wasconstructed (SEQ ID NO: 3; FIG. 58) and expressed as product (SEQ ID NO:4; FIG. 59).

Full length firefly luciferase[1-550] (SEQ ID NO:26, FIG. 62) and afusion protein containing N-terminal FFL [1-475] followed by a cleavableSNAP sequence [187-206] containing a polyhistidine tag was successfullycloned and expressed in E. coli (FIG. 24). Additionally, a fusionprotein containing N-terminal SNAP25[187-206] followed by full lengthFFL[1]550] was also designed, purified, and tested to determine whetherN-terminal SNAP-25 can alter FFL activity. Strong signals were obtainedfrom bacterial expressing the fusion proteinSNAP-25[187-206]-FFL[1-550], with signal doubling in intensity when inpresence of BoNT/A complex. Overlapping split FFL constructFFL[1-475]-SNAP25[187-206]-FFL[265-550] also produced a clear nearlytwo-fold signal increase in presence of BoNT/A. Overlapping split FFLconstruct FFL[1-478]-SNAP25[187-206]-FFL[265-550] (SEQ ID NO: 4) alsoproduced a significant increased signal in presence of BoNT/A. Theconstruct FFL[1-475]-SNAP25[187-206] produced an insignificant signalwhen in presence or absence of BoNT/A treatment.

FIG. 25 A and B provide a synthesis schematic and cloning strategy forrecombinant overlapping luciferase fragments having an interspacedSNAP25 sequence for BoNT/A detection. For other BoNT serotypes, thecorresponding sequences from the appropriate SNARE complex molecule areused.

Example 13 Expandable Bioluminescent Detection System

A dual-strategy approach was employed to create an expandablebioluminescent detection system for the detection of toxin or proteaseactivity. This system was initially developed for detection of BoNT/A asa model, and can readily expandable to detection of all BoNT classes andsubtypes as well as other toxins or enzymes having measurable activity.Strategy 1 comprises a dual reaction chamber including 2 vials and twotypes of beads (FIG. 26A). This strategy uses FFL fusion proteins torecombine and to restore FFL activity similar to previously describedmethods (Paulmurugan et al., “Combinatorial library screening fordeveloping an improved split-firefly luciferase fragment-assistedcomplementation system for studying protein-protein interactions,” AnalChem 79:2346-2353 (2007); Paulmurugan et al., “Firefly luciferase enzymefragment complementation for imaging in cells and living animals,” AnalChem 77:1295-1302 (2005)). The N-terminal region of FFL that is unableto support bioluminescent reactivity (residues 1 to 475 or shorter) wasfused to the binding domain of a known protein having a wellcharacterized binding affinity for another binding partner. The otherbinding partner was fused to the C-terminal portion of FFL (residues 476to 550) and to a BoNT/A cleavable SNAP25 sequence (FIG. 26 A1). Themodified C-terminal FFL fusion was attached to beads via the SNAP25domain and maintained in a macrofluidic reaction chamber that is capableof interfacing to 1 ml sized sample volumes. Interaction with BoNT/Acleaves the C-terminal FFL fusion, leaving the substrate on the beadsurface, to which it has a specific affinity (e.g. by use of ahistidine-tag) (FIG. 26 A2). Alternatively, the cleaved substrate can bebound on a specific enrichment column. After sufficient exposure to thesample, the accumulated cleaved substrate was eluted and transferred toa microfluidic reaction chamber where it encountered the immobilizedN-terminal FFL domain fusion protein. Combination of the FFL fragmentsoccurs through dimerization of the binding protein domains andbioluminescence is detected in the presence of adenosine triphosphate(ATP) and luciferin. The advantage of the dual chamber is thataccumulation of cleaved substrate can be obtained over time for samplesthat do not require further purification, such as, for example, clearserum samples. Turbid samples may require additional purification suchas by immuno-capture of the toxin, for which a single chamber (describedbelow) may be more suitable.

In Strategy 2, a single chamber system is employed wherein a luminogenicFFL derivative is directly exposed to affinity-enriched toxin such asBoNT/A (FIG. 26 B). The luminogenic FFL derivative can either beconstructed directly to the fusion protein or with a fusion ofoverlapping FFL fragments that are spaced by a cleavable SNAP25sequence. We have found that the overlapping FFL fragments (1-478) and(265-550) recombine to produce up to 4% of the activity of FFL, possiblyby formation of a heterodimer.

Example 14 Bioluminogenic Substrate Using a Bioluminescent Bead-BasedDetection System

A bioluminogenic BoNT/A peptide, FFL(1-550)SNAP(187-206)-TEVsite-His₆,(FFLSH), was developed and optimized to detect BoNT/A activity using abead-based bioluminescent detection system. The peptide is comprised ofa fusion protein containing full-length bioluminogenic fireflyluciferase (FFL) (amino acid residues 1-550), a SNAP-25 sequence (aminoacid residues 171-206) that can be cleaved by BoNT/A, a positive controlcleavage site, and an affinity tag. This fusion peptide was furtheroptimized to contain linker sequences on both sides of the SNAP-25sequence and a lysine-rich anchor sequence(FFL(1-550)-L1-SNAP(187-206)-L2-TEVsite-A-His₆ (FFL-L1 SL2TAH (SEQ IDNO: 29) (FIGS. 27 A and 61). These luminogenic peptide can berecombinantly expressed in E. coli and immobilized onto beads (FIG. 27B).

More specifically, the FFL-L1SL2TAH fusion protein was immobilized ontoCNBr activated Sepharose beads via an octa- or deca-lysine anchorpeptide (Anchor (K8+GLE): K,K,K,K,K,K,K,K,G,L,E (SEQ ID NO: 30) andAnchor (K10+GLE): K,K,K,K,K,K,K,K,K,K,G,L,E (SEQ ID NO:31)) and containsa polyhistidine tag (Histidine Tag (H6): H,H,H,H,H,H (SEQ ID NO: 32))for protein purification (FIG. 27 A; “A” and “H”, respectively; FIG.63). Additionally, a cleavage site that can be recognized by the TobaccoEtch Virus (TEV) protease (ENLYFQG (SEQ ID NO: 33)) was inserted as apositive control (FIG. 27 A; “T”; FIG. 63); the addition of thiscleavage site is optional. To optimize the turnover rate for the BoNT/Acatalysed proteolysis, a serine-glycine linker was introduced at theN-terminus of the SNAP25 peptide (Linker 1 (G4SG4): G,G,G,G,S,G,G,G,G(SEQ ID NO: 34)) and a glycine linker was introduced at the C-terminus(Linker 2(G6): G,G,G,G,G,G (SEQ ID NO: 35)) of the SNAP25 peptide (FIG.27 A; “L1” and “L2”, respectively; FIG. 63). This bioluminogenic FFLfusion protein can also serve as a soluble, non-immobilized substrate,with the inhibitory domain replacing the anchor peptide. Theincorporation of glycine rich linkers on both sides of the SNAP-25sequence (FFL-L1SL2TAH, FIG. 27, SEQ ID NO: 25, FIG. 61) resulted incleavage of the bioluminogenic FFL fusion protein at a ˜30-fold higherrate by BoNT/A light chain (LC) than the same substrate that lacked oneor both linkers.

In some embodiments the FFL-L1SL2TAH fusion protein linker regions maycontain additional glycine or serine residues. Additionally, theFFL-L1SL2TAH fusion protein may be immobilized onto Cyanogen-bromide(CNBr) activated Sepharose beads or nickel nitrilotriacetic acid(Ni-NTA) beads.

Example 15 Detection of BoNT Using Bioluminescent Fusion Proteins

Testing Fire Fly Luciferase Protein Substrates for BoNT Detection.

As previously described, recombinant luciferase proteins that becomeactivated by specific cleavage reactions mediated by the neurotoxin'smetalloprotease activity were genetically engineered. We havesuccessfully managed to clone and express the following BoNT/Asubstrate-firefly luciferase (FFL) fusion proteins and controls in E.coli:

FFL[1-475]SNAP25[187-203]-His₆,

FFL[1-550]-His tag (full length FFL, control),

SNAP25[187-203][FFL1-550]-His₆

FFL(1-550)SNAP(187-206)-TEVsite-His₆, (FFLSH), and

FFL(1-550)-L1-SNAP(187-206)-L2-TEVsite-A-His₆ (FFL-L1 SL2TAH (SEQ ID NO:25).

The luminescence signal of the full length recombinant FFL[1-550]reaches 16×10⁶ relative luminescence units (RLU) when combined withONE-Glo reagent (5′-fluoroluciferin containing, from Promega), whereasthe fusion of incomplete N-terminal FFL with the BoNT/A cleavablesubstrate sequence (FFL[1-475]SNAP25[187-203]-His₆) gave only a residualsignal of 25×10³ RLU, which was expected. SNAP25[187-203][FFL1-550]-His₆produced a signal of approximately 5×10⁶ RLU. It is anticipated thatFFL[1-475]SNAP25[187-203]-His₆ can be cleaved by BoNT/A after which itshould be able to reconstitute active FFL when combined with thec-terminal portion of FFL (residues 265 to 550), however, therecombinantly expressed c-terminal portion of FFL was unstable.Therefore, FFL(1-550)SNAP(187-206)-TEVsite-His₆ (FFLSH) was expressedand purified. FFLSH contains a BoNT/A cleavable site as well as aTEV-protease cleavable site for control experiments, followed by ahexahistidine tag (His₆) for bead-immobilization (FIG. 28 A). BoNT/Acleavage removes the tag from FFLSH, but not from FFL (control). TEVcleavage removes the tag from both FFL and FFLSH. Prior toBoNT/A-exposure, FFLSH generated the same bioluminescent light signalintensity as FFL (FIG. 28 B). After treatment with BoNT/A, the intensityof the cleaved FFLSH was 5.4-fold higher than that of the non-cleavableFFL control. This signal-to-noise difference was further enhanced bysubsequent addition of yttrium (III) salts, which quenched lightgeneration of uncleaved FFLSH and FFL in a H₆ tag-dependent manner. TheBoNT/A-cleaved and tag-free form of FFLSH produced 158-fold more lightthan BoNT-treated FFL that still contained the H₆ tag (FIG. 28 B). Thebioluminescence of FFLSH in solution, on nickel nitrilotriacetic acid(Ni-NTA) beads, with and without BoNT/A LC treatment was evaluated (FIG.29 A). As previously described, when this fusion protein was bound toNi-NTA beads, the FFL bioluminescence was substantially quenched whencompared to FFLSH in solution (FIG. 29 B). BoNT light chain (LC) Acleaves the FFLSH at the SNAP25-cleavage site for BoNT/A, resulting inincreased luminescence in the supernatant. The cleavage of FFLSH isdependent on the concentration of LC A (FIG. 29 C). Additionally, as thebioluminogenic substrate is based on the SNAP25 sequence, this substratecan be cleaved by BoNT serotypes A, C, and E, but not B (FIG. 30 A, B).

The mechanism by which Y (III) salt addition quenches the lightproduction of H₆-tagged luciferases was investigated. In presence of theH₆ tag and Y (III) ions, FFL and FFLSH form insoluble microaggregatesthat do not pass through PVDF-filters with pore-sizes smaller or equal0.45 μm (FIG. 31). The aggregates are unable to catalyze thebioluminescence reaction. Adsorption onto surfaces is similar toaggregate formation and has been shown to denature luciferase molecules(Hlady 1991).

Furthermore, H₆ and alternative tags were used to removenon-BoNT-cleaved FFLSH from the sample by affinity chromatography.Instead of using Y (III) ions to inhibit uncleaved FFL or FFLSH,uncleaved FFLSH was removed with chromatographic nickel nitrilotriaceticacid (Ni NTA) beads. This method was successfully for an FFLSH-basedALISSA prototype. A bead-bound BoNT/A from spiked human serum sampleswas reacted with FFLSH. Ni NTA beads were then added and the supernatantwas mixed with luciferase reaction buffer (ONE-Glo, Promega) containingATP, Mg²⁺ and 5-fluoroluciferin. Without any optimization of the FFLSHsubstrate, as little as 2.4 fmol BoNT/A complex in a 150 μL sample wasdetected (FIG. 32). However, the extra step of having to removeuncleaved FFLSH by addition of Ni NTA beads is inconvenient and addsextra time to the assay execution. The observed bioluminescent detectionsignals of BoNT-cleaved FFLSH were virtually free of background noise,which is a substantial advantage over ALISSA with fluorogenic peptides.

Detection of Dual-Step Enzyme Cascades with Bioluminescent Readout forUse in the ALISSA.

As previously described, the original BoNT/A ALISSA uses fluorogenicpeptide substrates for the detection of BoNT's enzymatic activity. Thesepeptide substrates are costly in production and contribute to asubstantial level of background fluorescence even when they are presentin their un-reacted, uncleaved form. Therefore, novel luminogenicsubstrates that produce a highly specific bioluminescent signal whenexposed to BoNT were produced. Such substrates consist of fusionproteins that are recombinantly expressed in E. coli, which make theirproduction inexpensive and efficient. The substrates contain aluciferase domain (firefly or renilla), a BoNT-cleavable domain, and aninhibitor of the bioluminescent activity of the intact fusion protein.In order to generate the appropriate inhibitor domain, mice wereimmunized with recombinant firefly luciferase (FFL), splenocytes wereselected from immune mice, and then fused to myeloma cells to producehybridoma cells that express FFL-inhibitory monoclonal antibodies (FIG.33).

A live-colony screening system for E. coli cells that produce FFL wastested and can be used to produce the luminogenic BoNT substrate. Usinga digital camera with a long time-exposure (>16 s), FFL-expressing E.coli bacteria on agar plates can be detected. This system may be used toscreen for fusion proteins that will contain inhibited (inactive) FFLand green fluorescent protein. Colonies that are fluorescent and onlyproduce a bioluminescent signal upon exposure to BoNT will encodeplasmid DNA for the desired substrate.

Example 16 Detection of Systemic BoNT

Detection of BoNT in Serum and Organs of Intoxicated Mice Using ALISSA.

Using the ALISSA method described herein, BoNT/A levels were measured inserum and organs of intoxicated mice. The ALISSA was performed on serum,lung and liver of mice that had been intraperitoneally injected withdifferent doses of BoNT/A complex or with a mock injection of bufferonly (control mice) (FIG. 34). Organs were homogenized using the Whirlbag method (Walsh et al., “Tissue homogenization with sterile reinforcedpolyethylene bags for quantitative culture of Candida albicans,” J. ClinMicrobiol. 25:931-932 (1987). BoNT/A was detected systemically as shownin FIG. 34. BoNT/A was detected in blood and liver harvested two hoursafter injection with toxin. BoNT/A levels in the lung remained low. Toapply existing and novel BoNT ALISSAs to measure toxin distribution inanimal sera and organs. In some embodiments BoNT ALISSAs may be used tomeasure the systemic content and tissue distribution in sub-lethallyBoNT-intoxicated mice. In certain embodiments the pharmacokinetic andserotype-dependent toxin distributions may be determined in organsfollowing parenteral or oral intoxication.

Detection of BoNT in the Blood of Animals in the Course of anIntoxication/Rescue Experiment.

The BoNT/A ALISSA method herein was used to determine the concentrationof BoNT/A in the blood of animals in the course of anintoxication/rescue experiment (FIG. 35). Briefly, mice were injectedwith lethal doses of BoNT/A, followed by injection of a rescue agentcontaining a mixture of monoclonal antibodies that neutralize BoNT/A.The ALISSA technique was used to directly measure the clearance ofBoNT/A from the circulation. While BoNT/A activity was clearly detected1 min. after injection of the rescue antibodies, most of it was removedfrom circulation within 30 min. The ALISSA antibody used in this studywas the original polyclonal anti-BoNT/A rabbit antibody from abcam thatbinds to different epitopes than the mAbs used for the rescueexperiment.

Example 17 Ultrasensitive Detection of BoNT and Anthrax Lethal Factor inBiological Samples by ALISSA

Abstract.

As previously described, both botulinum neurotoxins (BoNTs) and anthraxlethal factor, a component of anthrax toxin, exhibit zincmetalloprotease activity. The assay detailed here is capable ofquantitatively detecting these proteins by measuring their enzymaticfunctions with high sensitivity. The detection method encompasses twosteps: (1) specific target capture and enrichment and (2) cleavage of afluorogenic substrate by the immobilized active target, the extent ofwhich is quantitatively determined by differential fluorometry. Becausea critical ingredient for the target enrichment is an immobilizationmatrix made out of hundreds of thousands of microscopic, antibody-coatedbeads, this detection method is termed an assay with a largeimmuno-sorbent surface area (ALISSA). The binding and reaction surfacearea in the ALISSA is approximately 30-fold larger than in mostmicrotiter plate-based enzyme-linked immunosorbent assays (ELISAs).ALISSA reaches atto (10-18) to femto (10-15) molar sensitivities for thedetection of BoNT serotypes A and E and anthrax lethal factor. Inaddition, ALISSA provides high specificity in complex biologicalmatrices, such as serum and liquid foods, which may contain variousother proteases and hydrolytic enzymes. This methodology can potentiallybe expanded to many other enzyme targets by selecting appropriatefluorogenic substrates and capture antibodies. Important requirementsare that the enzyme remains active after being immobilized by thecapture antibody and that the substrate is specifically converted by theimmobilized enzyme target at a fast conversion rate. A detailed protocolto conduct ALISSA for the detection and quantification of BoNT serotypesA and E and anthrax lethal factor is described.

Introduction.

Botulinum neurotoxins (BoNTs) are considered the most potent toxinsknown. By extrapolation from primate studies, the lethal human dose is1-2 ng/kg body weight when intravenously injected (Gill 1982). SevenBoNT serotypes (A-G) are known to be produced by Gram-positive anaerobicbacteria of the genus Clostridium (Arnon 2001). BoNT/A and B (and tosome extent E and F) are the main etiological agents of human botulism(Long 2007). Infant, food-borne and wound botulism are its most commonforms (Koepke 2008; Werner 2000; Sobel 2004). Natural BoNT is producedas a 900-kDa complex that contains the 150-kDa holotoxin consisting of a50-kDa light and 100-kDa heavy chain, plus several nontoxicneurotoxin-associated proteins (NAPs) (Simpson 1981; Sakaguchi 1982).Once in the bloodstream, the BoNT holotoxin targets and enters motorneurons, inside which the toxin's light chain zinc metalloproteasesubunit hydrolyzes SNARE proteins (Volknandt 1995). BoNT cleaved SNAREproteins no longer mediate the fusion of acetylcholine-containingsynaptic vesicles with the terminal motor neuron membrane (Lalli 2003).This efficiently shuts down neurotransmitter release into theneuromuscular junction, leading to flaccid paralysis on the macroscopicscale. Each BoNT serotype cleaves one or more of the three SNAREproteins (SNAP25, VAMP, and syntaxin) at specific peptide bonds (Lalli2004; Schiavo 1993; Schiavo 1993; Schiavo 2000; Schiavo 1992).

BoNTs have gained popularity as cosmetic drugs in recent years, and havealso been successfully used for the treatment of a variety ofneurological and neuromuscular disorders (Schantz 1992; Johnson 1999).However, because of the lack of a standardized testing procedure, theunits of biological activity are often unable to be directly convertedinto precise doses for human use, and overtreatment with BoNTs can causeiatrogenic forms of botulism (Partikan 2007; Crowner 2007). BoNT is alsoa potential biothreat agent because of its extreme potency andlethality, its ease of production and transport, and the need forprolonged intensive care of intoxicated persons (Arnon 2001).

The clinical diagnosis of botulism requires the presence of the toxin bedemonstrated in a clinical specimen. The mouse bioassay is most commonlyused. For example, it is applied for the analysis of stool and enemasamples from suspected cases of infant botulism (CDC 1998; Schantz1978). Mice are intraperitoneally injected with a sterile filteredsample and observed for signs of botulism. Furthermore, neutralizingantibodies can be used to specify the serotype of the causative BoNT.The mouse bioassay has a detection limit of 10-20 pg of neurotoxin, andtypically requires up to 4 days turnaround time (CDC 1998). The assaydetailed here can detect BoNT/A and BoNT/E with atto (10-18) and femto(10-15) molar sensitivity, respectively, and requires only a fraction ofthe time of the mouse bioassay (2.5 h ALISSA for BoNT/A) (Bagyramyan2008).

Anthrax lethal factor (LF) is another zinc metalloprotease that has beensuccessfully been adapted for use in the ALISSA. LF constitutes one ofthe three components of anthrax toxin that is produced by Bacillusanthracis, together with protective antigen (PA) and edema factor (EF)(Brossier 2001). LF specifically cleaves members of themitogen-activated protein kinase kinase (MAPKK) family, leading to theinhibition of essential signaling pathways. LF alone is not toxic; itrequires the presence of PA for its translocation into cells (Brossier2001). Macrophages are believed to be primarily affected by LF (Hanna1993). A specific and sensitive assay for the detection of LF ispotentially useful for early diagnosis of anthrax infection and isexpected to be a useful research tool to advance the understanding ofthe mechanism of action of anthrax toxin (Boyer 2007).

Detection Principle.

The method described here is referred to as an assay with a largeimmuno-sorbent surface area (ALISSA) (Bagramyan 2008). ALISSA measuresthe specific proteolytic activity of BoNT/A, BoNT/E, or LF after itscapture and enrichment on a beaded immunoaffinity matrix that containstarget-specific antibodies. Antibodies were chosen such that the enzymeretains its catalytic activity after immobilization. Following theremoval of nontoxin-specific sample components through stringent washes,the toxin-specific enzyme activities are determined by measuring thecleavage of specific fluorogenic peptide substrates (Bagramyan 2008).Fluorogenic peptide substrates for BoNTs and LF are commerciallyavailable, or can be synthesized by classical solid-phase peptidechemistry (Schmidt 2003). The fluorogenic substrates contain afluorescence donor and acceptor pair. Fluorescence of the fluorophore isquenched via the Förster resonance energy transfer (FRET) effect whenthe donor and acceptor are in close proximity to each other (preferablyless than 10 nm) (Forster 1948). The toxin's proteolytic activityhydrolyzes a peptide bond that leads to the separation of donor andacceptor, thereby releasing the unquenched fluorescence of thefluorophore (FIG. 1B). In contrast to the classical FRET effect, theacceptors used here are nonfluorescent. Therefore, the difference in thefluorescence of the donor before and after enzymatic cleavage ismeasured in the ALISSA. The peptide substrates used here containN-terminal fluorescein labels, such as 5-carboxyfluorescein (5Fam) orfluorescein isothiocyanate (FITC) as donors with 4,4-dimethylaminoazobenzene-4¢-carboxylic acid (Dabcyl) as an acceptor near theC-terminus; or alternatively, they contain a donor/acceptor pair ofortho-aminobenzoic acid (o-Abz)/2,4-dinitrophenyl (dnp).

Another critical component of the ALISSA is the beaded immunomatrix. Itsimmunosorbent surface area is approximately 30-fold larger than thetypical microplate well surface of a classical enzyme-linkedimmunosorbent assay (ELISA), and at least 5 mg of antibody are used foreach data point of an ALISSA measurement (Bagramyan 2008). Thetarget-binding light chains of the antibodies are directed away from thebead surface and toward the sample solution by immobilization via theirheavy chains, the Fc regions, on protein NG-coated agarose beads.Bleeding of antibodies into the sample solution would have a detrimentaleffect on the assay's sensitivity. Therefore, the antibodies are loadedwell below the specified loading capacity of the beads and arecovalently conjugated with the beads by an irreversible chemicalcross-linker.

The ALISSA not only concentrates and preserves enzyme activities of theimmobilized toxin, but also has a profound effect on kinetic properties.Enzymatic turnover rates of ALISSA-immobilized enzymes are dramaticallyincreased over those of the reaction of nonimmobilized enzymes, leadingto a strong signal amplification effect on the order of billions ofcleaved substrate molecules per bound toxin molecule per hour (Bagramyan2008).

The ALISSA for BoNT/A was originally used to detect BoNT/A in spikedsamples of human serum, gelatin phosphate diluent (GPD, used in clinicaldiagnosis of infant botulism) and liquid foods, such as milk and carrotjuice (Bagramyan 2008). ALISSA's high sensitivity (attomolar detectionof BoNT/A) is accompanied by high target specificity and robustness.Nontoxin proteases of most sample types do not lead to false-positiveALISSA signals because they are removed in stringent wash steps (FIG.36A-D).

ALISSA has now been expanded to detect anthrax LF and BoNT serotype E,but the detection of many other targets seems feasible, provided thatsuitable substrates and antibodies can be obtained. Thus, ALISSA has thepotential to significantly improve the diagnosis of botulism, anthraxinfection and potentially other serious infections, and could serve toprotect humans in biomedical and biodefense scenarios.

Materials

Instruments.

A 100SD Microcentrifuge was used from USA Scientific, USA forcentrifugation. For more than five assays a microcentrifuge was used,such as Eppendorf Model 5417R, or a centrifuge that accommodated 15-mLconical tubes (e.g., Beckman Allegra 6R). A Microplate mixer (e.g.,Multi-Microplate Genie, Scientific Industries, Inc., USA) and Rotisseriefrom Labquake (Barnstead International, Dubuque, Iowa) were used formixing and rotating, respectively. A standard laboratory incubator (37°C.) was used for incubation purposes. A microtiter plate readingspectrofluorometer: e.g., Wallac 1420 Multilabel Counter Victor2(PerkinElmer) or SpectraMax M2 or better (Molecular Devices, USA) wasused to measure fluorescence. The Victor2 spectroflurometer performed atmuch higher sensitivity than the SpectraMax M2 instrument. However, theSpectraMax M2 could be manually set to any desired excitation/emissionwavelength in the UV/VIS spectrum, which was very helpful for initialassay design. In contrast, the Victor2 required a set of optical filterswith fixed transmission wavelengths.

Plastic Ware.

The plastic ware used in the experiments was sterile and protease free,and was preferably autoclaved. The following plastic ware materials wereused throughout the experiment: 2 mL microcentrifuge collection tubes;1.5 mL microcentrifuge sample tubes; 15 mL conical polypropylene tubes(BD, Falcon, USA); 0.5 mL and 2.0 mL Seal-Rite microcentrifuge tubes,amber (USA Scientific); 96-well 300-μL black microplates or 96-well150-μL black microplates (Whatman or Greiner, USA); luer-lock disposablesyringes: 1, 3, and 5 mL (Cole-Parmer, USA); and 5 mL and 1 mL Mobicolcolumns with 10-μm pore size for lower and upper filters (MoBiTec GmbH,Germany) or screw cap spin columns (Pierce, USA). Spin columns wererecycled for reuse by removing the upper filter and washing the columnmultiple (at least five) times in 70% ethanol and then in ultrapureprotease-free water (ten times). The columns were then dried andequipped with a new upper filter (MoBiTec, Germany).

Reagents.

BoNT serotypes A and E from Clostridium botulinum in the form of the900-kDa complex, 150-kDa holotoxin, or 50-kDa light chain were from ListBiological Laboratories or Metabiologics, Inc. (Madison, Wis.). In somecountries, the possession and laboratory use of BoNTs is regulated bylaw, and restrictions regarding permissible amounts and shipping exist.The catalytic BoNT light chain and anthrax LF are generally accepted asnontoxic. BoNTs should be handled with extreme caution and aerosolformation must be avoided. The use of a class II or III biologicalsafety cabinet was recommended for experimentation with BoNTs.Recombinant B. anthracis anthrax lethal factor was from List BiologicalLaboratories. Rabbit polyclonal antibody for C. botulinum A Toxoid,ab20641 was from Abcam (Cambridge, Mass.) and mouse monoclonal antibodyfor light chain of BoNT/A was a gift from Dr. Larry Stanker, USDepartment of Agriculture. Rabbit polyclonal antibody for C. botulinumtoxin serotypes E was from Metabiologics, Inc. (Madison, Wis.) and goatanti-lethal factor from B. anthracis was from List BiologicalLaboratories.

ALISSA Substrates.

The following substrates were from List Biological Laboratories:SNAPtide peptide substrate (FITC/Dabcyl) for C. botulinum neurotoxintype A, or SNAPtide (o-Abz/Dnp) peptide substrate for neurotoxin type A;SNAPtide, unquenched calibration peptides for SNAPtide peptide substratewith FITC/Dabcyl and o-Abz/Dnp; SNAP Etide (o-Abz/Dnp) peptide substratefor C. botulinum neurotoxin type E; and the MAPKKide peptide substrate(Dabcyl/FITC) for anthrax lethal factor.

As an alternative to FITC-labeled SNAPtide, it was also possible toobtain custom-synthesized peptides from various sources. A fluorogenicBoNT/A substrate was successfully used containing the followingsequence: 5Fam-TRIDEANQRATK(DABCYL) X-amide (#115, SEQ ID NO: 21), where5Fam is at the α-amino group, Dabcyl at the ε-amino group of lysine, andX is norleucine with an amide C-terminus. The 5Fam label was much morestable than the FITC-labeled N-terminus, leading to lower backgroundfluorescence.

Reagents and Buffer Conditions.

The following regents were used during the course of the experiment:deionized water (ultrapure) with 18 MO/cm or lower conductivity,protease-free, 0.2 μm filtered, and autoclaved;4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES acid);acetonitrile, CH₃CN (30%); zinc chloride (ZnCl₂); sodium chloride(NaCl); tween-20; dimethyl sulfoxide (DMSO); ethylenediaminetetraaceticacid (EDTA), 20 mM, pH 8.2; disuccinimidyl suberate (DSS), No-Weighformat, M.W. 368.35 g/mol, spacer arm 11.4 Å, 8×2 mg vials (Pierce,USA); pooled human serum (Sigma or Innovative Research, USA); HEPESpotassium salt; and immobilized protein NG Plus, 50% slurry (Pierce,USA) or immobilized protein NG Plus, 25% slurry (Santa CruzBiotechnology, Inc., USA).

The following buffers conditions were used: Coupling buffer (10×): 100mM sodium phosphate, 1.5 M NaCl, pH 7.2; Immunoprecipitation (IP)/washbuffer: 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 (NP-40, USBiological, USA), 5% glycerol, pH 7.4; Conditioning buffer (100×):neutral pH buffer (Pierce Crosslink Immunoprecipitation Kit, Pierce,USA); Elution buffer: pH 2.8 (Crosslink Immunoprecipitation Kit, Pierce,USA), or 100 mM glycine HCl buffer, pH 2.8, or from Gentle Ag/Ab Bindingand Elution Buffer Kit (Pierce, USA); Reconstitution and reaction bufferfor BoNT/A light chain: 20 mM HEPES, pH 8.2, 0.5 mg/mL bovine serumalbumin (BSA) or 0.1% Tween-20; Reconstitution buffer for BoNT/Aholotoxin: 20 mM HEPES, pH 8.0, 2.5 mM DTT, 0.3 mM ZnCl₂, and 1.0 mg/mLBSA; Reaction buffer for BoNT/A holotoxin and complex: 20 mM HEPES, 0.3mM, ZnCl₂, 2.5 mM DTT, and 0.1% Tween-20, pH 8.0; Reconstitution bufferfor BoNT/E: 50 mM HEPES, pH 7.8, 0.1% Tween-20; Reaction buffer forBoNT/E: 50 mM HEPES, pH 7.8, 200 mM NaCl, 0.1% Tween-20; andReconstitution and reaction buffer for anthrax LF: 20 mM HEPES, pH 7.2,125 μg/mL BSA. The quenched SNAPtides, SNAP Etide, and MAPKKide wereprepared as 2.5 mM stock solutions and the unquenched calibrationpeptides as 1 μM stocks, all in DMSO. Stock solutions of reconstitutedfluorogenic substrates were kept at −20° C. in small aliquots.Unnecessary freeze/thaw cycles were avoided. Dry peptide powders werestored in a desiccator in the cold. All fluorogenic substrates were keptin the dark.

Methods

Preparation of the Immunomatrix.

Binding of Antibody to Protein A/G Plus Agarose: The following protocolwas adapted from the Pierce Crosslink Immunoprecipitation (IP) protocol(Pierce, Rockford, Ill., USA) with some additional modifications. Thisprotocol was designed to yield enough antibody-coated beads for tenassay reactions (5 μg antibody per reaction and data point). It isrecommended that the ALISSA be conducted at least in duplicate (tworeactions per sample). The procedure can be proportionally scaled toprepare immunomatrix for up to 20 assay reactions using the same plasticware and column sizes.

Ten mL of coupling buffer (1×) was prepared for each IP reaction bydiluting the coupling buffer (10×) with ultrapure water. The protein NGplus agarose beads were evenly suspended by gently swirling the bottle.Using a pipettor equipped with a cut pipette tip, 100 μL of thesuspended Pierce resin slurry or, alternatively, 200 μL of the SantaCruz resin slurry per reaction was added to a 5-mL Mobicol column(approximately 0.5 mm was cut off the plastic tip of a 300-μL Raininpipette using a sterile single-sided razor blade or scalpel to createthe cut pipette tip). The column was placed into a 15-mL conicalpolypropylene tube. The Luer-Lock cap was attached to the Mobicol columnand connected to a 5-mL air-filled disposable syringe. The plunger wasgently pushed into the syringe until all liquid was removed from thecolumn. Alternatively, the column containing resin was centrifuged for 1minute at approximately 1,000×g at 22° C., and flow-through wasdiscarded. The resin in the column was washed by adding 1.0 mL ofcoupling buffer (1×) and flow-through was discarded using the syringetechnique as previously described. This wash was repeated one time.

In a separate microcentrifuge tube, coupling buffer (10×), water, and 50μg of antibody in solution was mixed to yield a final volume of 500 μLof antibody solution at a dilution of coupling buffer (1×). Thisprotocol was optimized for 5 μg of antibody per enzymatic reaction (onedata point). Depending on the number of assays, amount of antibody,resin, cross-linker, and buffer volumes were proportionally scaled. Forexample, antibody received at a concentration of 1 μg/μL was mixed with50 μL of coupling buffer (10×), 400 μL water and combined with 50 μL ofthe antibody in solution.

After the wash, the bottom of the column was gently tapped on a papertowel to remove any excess liquid. The bottom plug was inserted to blockthe column's drain. The antibody solution prepared as previouslydescribed was immediately transferred into the resin-containing column.The beads were never allowed to dry. The closing screw cap was attachedto the column and the column was incubated on the rotating rotisserie at22° C. for 1 hour. The slurry remained suspended at all times duringincubation. The bottom plug and cap was removed and saved. The columnwas placed into a 15-mL conical collection tube and liquid was removedfrom the column as previously described (using pressure orcentrifugation). The flow-through was saved to verify antibody couplingusing the Bradford protein quantification assay (Bio Rad, USA) or thebicinchoninic acid (BCA) protein assay (Pierce, USA). The resin waswashed once with 0.5 mL and then twice with 1.5 mL of coupling buffer(1×). Flow-through was removed and discarded as previously described.

Cross-Linking of the Protein A/G-Coupled Antibody.

To prepare a fresh 25-mM DSS solution, 217 μL DMSO was added into a newvial of 2 mg DSS, by inserting the pipette tip through the vial's foilcovering. The solution was thoroughly mixed by pipette aspiration anddispensing multiple times until all DSS was fully dissolved. In a newmicrofuge tube, the 25 mM DSS solution was further diluted 1:10 withDMSO to yield 2.5 mM DSS. The bottom of the column was tapped withantibody-coated resin prepared as previously described on a paper towelto remove excess liquid. The bottom plug was inserted. Two hundred andfive μL of coupling buffer (1×) was added to the antibody-coated resinand resuspended. To obtain a final concentration of 450 μM DSS, thisresin solution was then combined with 45 μL of the 2.5 mM DSS solutionprepared as previously described. The column was capped with a closingscrew cap. The cross-linking reaction was incubated for 1 hour at 22° C.on the rotisserie. The bottom cap and plug were removed and saved. Thecolumn was placed in a collection tube and flow-through was drained aspreviously described. Two hundred and fifty μL of elution buffer wasadded to the column the liquid was drained gently, as previouslydescribed. The flow-through was saved to verify antibody cross-linkingby measuring protein concentration as previously described. The columnwas washed twice with 0.5 mL of elution buffer to remove non-crosslinkedantibody and unreacted DSS. Next, the column was washed twice with 1.0mL of ice-cold IP/wash buffer and the liquid was drained after eachwash. 0.5 mL of IP/wash buffer was added and the column with resin wastransferred into a new tube. Fifty μL of this final resin suspensioncontained beads with approximately 5 μg antibody.

Storage Conditions.

The resin with the cross-linked antibody could be stored for up to 5days in IP/wash buffer at 4° C. The antibody-bound resin was transferredinto a new, protease-free microcentrifuge tube. For longer storage(maximum of 2 weeks at 4° C.), resin was stored in coupling buffer (1×)and the tube was wrapped with parafilm.

ALISSA for BoNT and LF

The following protocol below describes the amounts and volumes used fora single ALISSA reaction (one data point). Each reaction was conductedin a separate tube. The number of tubes can be scaled according to thenumber of samples to be analyzed.

Toxin Enrichment and Immobilization.

The sample to be analyzed (e.g., serum and liquid food) was combinedwith IP/wash buffer in a 3:1 ratio. The final volume of sample mixedwith IP/wash buffer ranged from 0.5 to 5 mL. Fifteen mL conical steriletubes were used for sample volumes larger than 1 mL and microcentrifugetubes were used for samples less than 1 mL. Fifty μL of resin withcross-linked antibody (prepared as previously described) was added tothe sample/IP/wash buffer. This solution was mixed and was incubated bygentle mixing on the rotisserie for 1-2 hours at 22° C. or 16 hours at4° C. A syringe was used to transfer the resin-containing sample into aspin cup column (Mobicol 1 mL or Pierce 1 mL spin column) with Luer-Lockconnector and placed into a collection tube (FIG. 37 a). Liquid wasdiscarded. Sample volumes larger than 1 mL were also processed using5-mL Mobicol columns with filter inserts and liquid was removed bycentrifugation. Two hundred mL of IP/wash buffer were added to the resinand centrifuged with spin column placed into collection tube (FIG. 37b). Flow-through was discarded. An alternative buffer, 20 mM HEPES, pH7.5, with 0.1% Tween-20, was used for BoNT assays. This process wasrepeated five times. Next, resin was washed once with 100 μL ofconditioning buffer (1×), once with 100 μL of 2.5 M NaCl, and twice with200 μL of ultrapure protease-free water. Resin was then resuspended in100 μL of ultrapure protease-free water, and transferred into a newmicrocentrifuge tube using a cut pipette tip. Finally, resin was wrappedwith parafilm and stored at 4° C. until used (up to 5 days).

Reaction of the Fluorogenic Peptide Substrate.

Each sample for the ALISSA required 10 μL of a prediluted 250 μMsubstrate stock, and the final concentration of the fluorogenicsubstrate in the reaction buffer was 5 μM. Thus, the 2.5 mM peptidestock solution of the appropriate fluorogenic peptide substrate wasdiluted with 30% CH₃CN in ultrapure protease-free water to a finalconcentration of 250 μM. The reaction buffer was prepared by dilutingthe prediluted fluorogenic peptide stock 50-fold in the appropriatetoxin reaction buffer. Each sample required 450 μL of reaction buffer.In a 2.0-mL amber microcentrifuge tube, 50 μL of resin of theimmobilized toxin sample was combined with 450 μL of thesubstrate-containing reaction buffer. A sample of 450 μLsubstrate-containing reaction buffer with 50 μL toxin-free resin wasincluded as a control. This control was used to establish the baselineof the fluorescence background. Each sample was incubated 1-3 hours bygently rotating the amber reaction tube(s) on the rotisserie inside anincubator at 37° C. The o-Abz-conjugated peptides react slowly;therefore, these reactions were prolonged for up to 16 hours. Twohundred and twenty microliters of the reaction mixture (beads included)were transferred into a well of a 96-well black microplate (300 μL wellvolume).

Fluorescence was measured with a Wallac 1420 Multilabel Counter Victor2spectrofluorometer or comparable plate reader. Excitation wavelengths ofλ_(ex)=485 nm and λ_(ex)=535 nm were used for BoNT and LF reactions withFITC/Dabcyl-peptides, respectively, and excitation wavelengths ofλ_(ex)=321 nm and λ_(ex)=418 nm were used for o-Abz/Dnp-peptide,respectively. The fluorescence intensity of the baseline (toxin-freecontrol) was subtracted from the fluorescence intensity of each of themeasured samples.

Calibration Curve.

The following protocol was adapted from the List Biological LaboratoriesStandard Curve protocol (Campbell, Calif., USA) and contains someadditional modifications. Calibration peptides that correspond to thecleaved, fluorescent substrate product were used to generate acalibration curve that allowed conversion of relative fluorescence units(RFU) into molar units of cleaved substrate. This value was also used tocalculate specific enzymatic activity, when the reaction time, volume,and protein amount were also recorded. (Bagramyan, K., et al., 2008). A0.5-mM stock solution was prepared by dissolving 1 vial of calibrationpeptide (˜49.4 nmol) in 98.8 μL of DMSO. The calibration peptide wasfurther diluted to a 1.0-μM solution by adding 5 μL of the 0.5 mMcalibration peptide stock solution to 2,495 μL of toxin reaction buffer.Each dilution was performed in triplicate using 220 μL/well for a 300-μLwell volume (96-well black microplate) or 120 μL of total reactionbuffer for a 150-μL well volume (reduced volume 96-well blackmicroplate). The dilution series was prepared as provided in FIG. 38.

The ALISSA assay was performed using BoNT/A, E and anthrax LF usingprotein G and A in a bead free assay. As shown in FIG. 36D, the ALISSAtechnology is applicable for use with anthrax LF and reaches femtomolarsensitivities (FIG. 36 d). ALISSA has now been expanded to detectanthrax LF and BoNT serotype E, but the detection of many other targetsseems feasible, provided that suitable antibodies and substrates can beobtained. Thus, ALISSA has the potential to significantly improve thediagnosis of botulism, anthrax infection and potentially other seriousinfections, and could serve to protect humans in biomedical andbiodefense scenarios.

Example 18 ALISSA Technology on Additional Targets

The ALISSA technology is also applicable for use with targets such asenzymes or toxins other than BoNT. The ALISSA technology was alsoextended to detection of human chitinases (e.g. CHIT1 and AMCase) (FIG.39) and non-metalloproteases (Pep1 and Pep2 of Aspergillus fumigatus)(FIG. 40A and B). As shown in FIGS. 39 and 40 a-b, the ALISSA technologyis applicable for use with a wide variety of targets including non-toxintargets and enzymes.

Example 19 ALISSA Technology Using BoNT Holoenzymes, Complexes, andLight Chains

Materials

Different antibodies that recognize epitopes on BoNT or BoNT lightchains (BoNT LC) were used to prepare the bead-based immuno-affinitymatrix for the ALISSA. The monoclonal antibody, 5A20.4, whichspecifically recognizes the BoNT/A LC, and the monoclonal antibody,2B24, which specifically recognizes the BoNT/B light chain (BoNT/B LC),were both received from J. Marks, Department of Anesthesia andPerioperative Care, University of California, San Francisco, Calif.,USA. The camelid heavy-chain-only antibodies V_(H) (VHH) that recognizeBoNT/A and BoNT/B were received from C. Shoemaker, Tufts School ofVeterinary Medicine, North Grafton, Mass. Protein NG PLUS-agarose wasfrom Santa Cruz Biotechnology, Inc. (cat. #sc-2003), the PierceCrosslink Immunoprecipitation Kit was from Pierce (cat. #26147),Cyanogen bromide-activated Sepharose 4B was from Sigma (CNBr-activatedsepharose, cat. #C9142), and wash spin cups were from MoBiTec (Mobicols,10 μm filter, cat. #M1002S).

Preparation of Beads Coupled with Anti-BoNT and Anti-BoNT Light ChainAntibodies

Antibodies that recognize BoNT/A and BoNT/B (camelid heavy chainantibodies), BoNT/A light chains (BoNT/A LC) (5A20.4 monoclonalantibody), and BoNT/B LCs (2B24) were coupled to CNBr-activatedSepharose beads for use in the bead-based ALISSA. Briefly, 660 mg ofCNBr-activated Sepharose beads were weighed and transferred into adisposable polysterene column washed with autoclaved H₂O. The beads wereswelled for approximately 15 minutes at room temperature using ice-cold1 mM HCl that was prepared by diluting 100 μl 10M HCl in 1 liter H₂O.The beads were subsequently washed with 200 ml of 1 mM HCl, drained, andcapped. Two mg of each antibody was prepared by adjusting the volume ofantibody with 2 mL of coupling buffer containing 0.1 M NaHCO₃ (sodiumbicarbonate) and 0.5 M NaCl, pH 8.3-8.5 (pH was adjusted with 1 M NaOH).The antibody solution obtained in the previous step was transferred ontothe column with CNBr-activated swollen beads. A portion of antibodysolution was saved to verify antibody coupling. An additional 4 mL ofcoupling buffer was added onto the column with antibody andCNBr-activated beads. The column was rotated gently at room temperaturefor 2-3 hours or at 4° C. overnight ensuring that the beads remainedsuspended during incubation. Flow-through was saved to verify antibodycoupling. The excess antibody was washed away with 10 gel volumes ofcoupling buffer. The remaining active groups were blocked with 0.3 MEthanolamine, pH 8.0 (+/−0.05) (active groups can also be blocked with0.3 M Tris pH 8.0). The beads were gently re-suspended and placed on arotator for 2 hours at room temperature (25° C.) or overnight at 4° C.The beads were then washed with 10 gel volumes at least three times withbuffers of alternating pH. Specifically, the beads were first washedwith basic coupling buffer (0.1 M NaHCO₃, 0.5 M NaCl, pH 8.3-8.5),followed by a wash with 0.1 M CH₃COONa (sodium acetate), and lastly, awash of 0.5 M NaCl, pH 4.0 (adjust pH with 10M HCl). Finally, the beadswere washed with 10 ml 1×PBS (Dulbbelco's PBS, Mediatech cat.#21-031-CM). Antibody coupled beads that were used immediately werere-suspended in 1×PBS, placed on ice to settle, and adjusted to a finalvolume for a 50% suspension. Antibody coupled beads that were not usedimmediately were re-suspended in 100 mM ammonium bicarbonate,lyophilized, and stored at 4° C.

Immunocapturing of BoNT Holotoxin, Complex and BoNT Light Chains

An immunocapture method was utilized to capture the BoNT/A and BoNT/Bholotoxins, complexes and light chains (LCs) using antibody coupledbeads that were prepared as previously described. A standard curve wascreated by preparing serial dilutions of each enzyme including BoNT/Aholotoxin, BoNT/A complex, BoNT/A LC (recombinant form), BoNT/Bholotoxin, BoNT/B complex, and BoNT/B LC (recombinant form). One tubewas included in each series for a toxin negative control. Two hundred μLof antibody-coupled beads were transferred into each tube of thestandard curve dilution series, including the toxin-negative control.The antibody-bound beads and enzymes were gently rotated for 4-5 hoursat room temperature or 1 hour at room temperature followed by overnightrotation at 4° C. on a rottiserie. Beads were pelleted by centrifugationfor 1 minute (1000×g). Spin cups were washed once with 500 μLprotease-free water before use. Five hundred μL aliquots of eachserum/bead mixture were centrifuged through the spin cups until theentire sample was processed. The beads were then washed with a series ofice-cold buffers including two washes with Immunoprecipitation (IP)Binding Buffer/Wash Buffer (0.025M Tris, 0.15M NaCl, 1% NP-40, 5%glycerol), one optional wash with Conditioning Buffer (Pierce, cat.#26147), and three washes with protease-free water. Beads werere-suspended in H₂O (2×100 μL for duplicate reactions or 3×100 μL fortriplicate reactions) and transferred to an Eppendorf tube.

Enzymatic Reaction with Immuocaptured and Immobilized BoNTs

The enzymatic activity of immobilized BoNT/A and BoNT/B holotoxins,complexes, and light chains (LCs) was determined by cleavage ofpreviously described BoNT/A cleavable fluorogenic substrates (#115 (SEQID NO: 21) and #116 (SEQ ID: NO 22). In order to reduce and pre-activatethe enzymes, the BoNT/A and BoNT/B holotoxins and complexes werepre-incubated with DTT (the BoNT/A LC and BoNT/B LC do not requirepre-incubation with DTT). For each replicate (BoNT/A holoenzyme andcomplex, BoNT/B holoenzyme and complex), including the toxin negativecontrol, 100 μL of antibody-bound beads with immobilized enzymes(prepared as previously described above in Immunocapturing of BoNTs andBoNT Light Chains) was transferred into 250 μL of BoNT/A reaction buffercontaining 20 mM HEPES, pH 7.4-7.5, 20 μM ZnSO4, 0.1% Tween-20, 10 mMDTT. For the bead negative control, 100 μL of water was transferred intothe reaction buffer. Samples were incubated for 20-30 minutes at roomtemperature. A solution of 250 μl of BoNT reaction buffer without DTTcontaining a final concentration of 12 μM of BoNT-cleavable peptides andnon-cleavable peptides was prepared. Solutions were prepared for theBoNT/A 5-FAM and 4-MU cleavable (#115 (SEQ ID NO: 21) and #116 (SEQ IDNO: 22), respectively) and control peptides (#112 (SEQ ID NO: 19) and#113 (SEQ ID NO: 5)) and the 5-FAM BoNT/B cleavable peptide (SEQ ID NO:24) for each replicate. Briefly, a 1:10 dilution of peptide was preparedfrom a 2.5 mM DMSO stock (stored at −20° C.) in 30% CH₃CN (acetonitrile)and then transferred into BoNT Reaction Buffer. Two-hundred fifty μL ofpeptide-containing buffer was distributed into each tube containingBoNT-bound antibody-bound beads. The final concentration of peptide was5 μM.

For BoNT/A and BoNT/B LCs, 100 μL of antibody-bound beads withimmobilized enzymes (prepared as previously described above inImmunocapturing of BoNTs and BoNT Light Chains) was transferred into 500μL of BoNT reaction buffer containing 20 mM HEPES, pH 7.6, 20 μM ZnSO4,0.1% Tween-20 and 1% BSA and 6 μM of BoNT/A 5-FAM and 4-MU cleavable(#115 (SEQ ID NO: 21) and #116 (SEQ ID NO: 22), respectively) andcontrol peptides (#112 (SEQ ID NO: 19) and #113 (SEQ ID NO: 5)),respectively).

Eppendorf tubes containing the antibody-bound beads with immobilizedenzymes and peptides were transferred in a carton box (protected fromlight), placed on a rotary shaker, and left at room temperatureovernight. The following day, tubes were centrifuged for 1 minute(1000×g) to re-pellet beads. Approximately 250-275 μL of solution wastransferred into each well of a black 96-well plate (Nalgene (Nunc,Fisher, cat. #12-566-09)) starting from the lowest concentration ofBoNT. Each fluorescence was measured twice, once at Ex 485 nm/Em 535 nmfor the 5-FAM peptides, and then at Ex 355 nm/Em 460 nm, for the 4-MUpeptides.

Example 20 BoNT/A ALISSA in a Systemic Mouse Model of Botulism

The pharmacokinetics of BoNT/A intoxication in mice were studied usingthe ALISSA approach described herein. Serum samples from intravenous(i.v.) and intragastric (i.g.) intoxication models were provided by Dr.Luisa W. Cheng (United States Department of Agriculture, Albany,Calif.). 5A20.4 monoclonal anti-BoNT/A light chain antibody-coupledCNBr-activated sepharose beads were used in the ALISSAs to captureBoNT/A. (FIGS. 41-42). Serum samples from intoxicated mice were analyzedby ALISSA using two sets of BoNT-cleavable fluorogenic peptidesubstrates and BoNT non-cleavable controls, which provided a method toqualitatively distinguish between BoNT and non-BoNT related proteaseactivities. The BoNT/A cleavable peptides and control peptides werefluorescently labelled with either 5-carboxyfluorescein (5-FAM) or4-Methylumbelliferone (4-MU) conjugated to their α-amino group and adark quencher, DABCYL, conjugated near their C-terminus. Each peptidesubstrate was combined with a BoNT/A1 non-cleavable control peptide thatwas conjugated with the respective other fluorophore. For example, a5-FAM-labeled control was combined with a 4-MU-labeled substrate andvice versa (control peptide #112 (5-FAM; SEQ ID NO:19) was used incombination with BoNT/A cleavable peptide #116 (4-MU; SEQ ID NO: 22),while control peptide #113 (4-MU; SEQ ID NO: 5), was used in combinationwith BoNT/A cleavable peptide #115 (5-FAM; SEQ ID NO: 21)) (FIG. 42).The difference in fluorescence intensities corresponded to the realsignal produced by BoNT/A only. Two different standard curves withBoNT/A cleavable and control peptides were generated in parallel withthe same antibody in pooled normal (non-intoxicated) mouse serum spikedwith BoNT/A1 complex. These standard curves were used to convert therelative fluorescent unit numbers into BoNT/A molar concentrations (FIG.43A and B). The ALISSA results from different combinations of BoNTcleavable and control substrates produced comparable results. BoNT/Aconcentrations in the tail blood of intoxicated mice were measured atdifferent time points after i.v. and i.g. delivery of low quantities ofBoNT/A1 complex. I.v. delivery of different concentrations of BoNT/Acomplex (0, 4 pg, 20 pg and 100 pg BoNT/A complex per mouse) werefollowed for one hour and i.v. delivery of 100 pg BoNT/A per mouse wasfollowed over time (FIG. 44 (Group I and II, respectively), FIG. 45 Aand B). Additionally, i.g. delivery of 4 μg BoNT/A complex per mouse wasfollowed over time (FIG. 44 (Group III)).

ALISSA detected approximately 7 to 13 aM BoNT/A in the mouse serum onehour after i.v. injection with 4 pg BoNT/A (n=4). A time course analysiswith 100 pg BoNT/A/mouse (n=3) showed femtomolar toxin concentrationsdetected in mouse serum after only one hour post i.v. injection (FIG. 45B). However, no toxin was detected at later time points (FIG. 45 B andC, >3 hours), which indicates that the systemic toxin is rapidlyabsorbed, most likely due to neural absorption.

In contrast, in the i.g. intoxication model, BoNT was slowly releasedinto the system as substantial time was required for the toxin to reachthe blood stream. BoNT/A serum levels were not detectable one hour afteri.g. intoxication with 4 μg BoNT/A complex per mouse. The BoNT/A serumconcentrations rose slowly and were 165-235 aM at two hours, and 647-723aM at seven hours after i.g. delivery of the toxin (FIG. 46 A and B).These results indicate that systemic BoNT/A becomes detectable 5-7 hoursafter intoxication, manifested by the presence of 18-38 aM of toxin inmouse serum. Twenty-four hours after intoxication, most BoNT/A waslikely absorbed by nerve endings, resulting in undetectable levels oftoxin. Most of the animals did not survive after more than 48 hoursafter intoxication.

The pharmacokinetic measurements of BoNT were further tested in anadditional i.g. intoxication mouse model. Mice (n=3) were orallyintoxicated with 1 μg BoNT/A1 complex and BoNT/A concentrations in thetail blood of intoxicated mice were measured at different time points(2, 5, 7, 8, 24, and 48 hours after toxin delivery, FIG. 46 C). SystemicBoNT/A1 became detectable in serum between 5 to 8 hours after gavage,leading to a spike at 7 hours with a serum equivalent concentration of18-38 aM BoNT/A1. Twenty-four hours after intoxication, most BoNT/A1 wasremoved from the blood (FIG. 46 C). The transition of BoNT from thegastric tract into the bloodstream was the rate limiting step in thei.g. intoxication model.

The ALISSA approach allowed real-time quantification of BoNT/A1 in serumof i.v., i.g. and orally intoxicated mice by demonstrating atto- tofemtomolar blood toxin concentrations. This assay demonstrates thatBoNT/A ALISSA would be a useful tool to conduct pharmacokinetic studiesof BoNT in humans with botulism or in patients who receive BoNT-basedmedical treatments.

Example 21 Quantification of BoNT in Intoxicated Neurons

ALISSA was used for the precise quantification of BoNT in intoxicatedneurons. Primary neuronal cells from 18-day old fetal rat hippocampiwere cultured for 14 days after seeding into neurobasal mediumsupplemented with 200 mM GLUTAMAX-I and 50× B27 (Invitrogen). Neuronalcells were exposed to 0, 2, or 20 nM BoNT/A in the presence of 55 mM KCLfor 30 minutes and then thoroughly washed. Neurons were plated ontocoverslips coated with poly-D-lysine (30 g/ml) and laminin (2 g/ml) at adensity of 75,000 per coverslip and shipped to City of Hope, Duarte,Calif. Monoclonal anti-BoNT/A light chain antibody (5A20.4) from theUniversity of California, San Francisco was used in the ALISSA. BoNT wasextracted using a lysis buffer containing: 0.025M Tris, 0.15M NaCl, 1%NP-40, and 5% glycerol (FIG. 47).

A number of different experiments with BonT/A1-intoxicated rathippocampal primary neuronal cells were performed using the ALISSAapproach. BoNT/A1 standards with recombinant BoNT/A light chain or BoNTcomplex were generated using 20 nM toxin to test for the presence oftoxin in high K+ medium with 20 nM BoNT/A1 (FIG. 48 A and B). The secondstandard, containing low (nM to aM) concentrations of BoNT/A1 complexspiked into the cell lysis buffer, was prepared to quantify the amountof BoNT/absorbed by neuronal cells (FIG. 49 A). The ALISSAquantification of BoNT/A was performed in both cytosolic and pelletfractions, and compared to standard curves prepared with serialdilutions of BoNT/A holotoxin (FIG. 49 B). Very mild conditions wereused to extract the toxin from the neurons. BoNT-treated neurons weredetached and collected from coverslips/wells by application of the lysisbuffer. The detached and partially lysed neurons were shock frozen inliquid nitrogen and shipped on dry ice from UCSF to City of Hope in ablinded format. ALISSA was performed on the neuronal extracts, thesupernatant of the BoNT-containing medium, and the wash buffer that wasused to wash the cells after co-incubation with BoNT (FIG. 49 C). Whenincubated with BoNT at 2 nM concentration, 75,000 cells still retained270 attomol (270e-18 mol) BoNT, corresponding to 2,167 active moleculesper cell (FIG. 49 C). The results from this assay represent the firstquantitative measurements of active BoNT molecules in intoxicated cells.In certain embodiments the ALISSA is conducted with Neuro2A and M17cells in vitro as well as with isolated neurons.

Example 22 BoNT/A ALISSA with Sera from Adult Botulism Patients

Patient serum, obtained from uninfected adults or adult patientsinfected with food-borne or wound botulism, was tested using BoNT/AALISSA. A BoNT/A cleavable substrate (#115 (SEQ ID NO: 21), black bar))was used to detect the presence of BoNT/A and a non-cleavable peptide(#112 (SEQ ID NO: 19), grey bar) was used as a control (FIG. 50). BoNT/Awas detected in serum samples from well characterized clinical cases ofBoNT/A food-borne botulism and wound botulism, but not in pooled humanserum. This represents the first BoNT/A ALISSA using sera from adultbotulism patients. These exemplary results demonstrate that ALISSA isparticularly useful for detection of botulism in clinical specimens.

Example 23 BoNT/A ALISSA with Infant Botulism Patient Serum

BoNT/A ALISSA measurements were performed using a serum sample of a wellcharacterized clinical case of BoNT/A infant botulism (IB). The samplewas provided by Dr. Stephen Arnon's laboratory at the CaliforniaDepartment of Public Health (C.D.P.H.) under CDPH-IB approval. BoNT/Aactivity was measured using a BoNT/A cleavable peptide substrate (#115(SEQ ID NO: 21), green bar)) and a control peptide (#112 (SEQ ID NO: 19)(FIG. 51 A). The differential fluorescence signal was converted intomolar BoNT/A concentrations using a standard curve (FIG. 51 B) and foundto be approximately 0.3 pg/mL holotoxin. This corresponds to aconcentration of approximately 1.8 pg/mL BoNT/A 900-kDa complex, whichis 44 times lower than the detection limit of the mouse bioassay. Thisrepresents the first BoNT/A ALISSA performed using sera from infantbotulism patients. These results illustrate the exemplary specificity ofthe ALISSA compared with the “gold standard” life mouse assay used todetect botulism, which demonstrates how the ALISSA is useful as adiagnostic tool and will improve public health by being directlyapplicable to naturally occurring botulism such as infant botulism. Incertain embodiments a reproducible ALISSA platform may be used to testserum samples from human cases of botulism. In some embodiments, the useof the ALISSA may be used regulatory approved for clinical diagnosticuse.

Example 24 Reagents for Microcolumn-Capture of BoNT Serotypes A and B(Including Subtypes)

Single Chain Antibodies (VHH) for BoNT ALISSA.

Microcolumn-enrichment of BoNT requires affinity reagents that 1) can bedirected and covalently attached to the column material to avoidbleeding of the reagent; 2) bind to the light chain (LC) of BoNTswithout inhibition of its enzymatic activity; 3) have a very highaffinity and specificity for a desired BoNT serotype. Although theoriginal ALISSA was based on antibodies, such substances are notnecessarily required as affinity reagents for microcolumns. Fusionproteins that contain antigen-binding fragments like scFvs or thebinding domains of single chain antibodies can be used instead and havethe advantage that they can also be engineered to accommodate a robuston-column immobilization chemistry and multivalent epitope recognition.However, the use of scFvs can be less efficient that other types ofantibodies, for example single chain antibodies from camels orcamel-like animals (camelids). Therefore, recombinant alpaca singlechain antibodies (VHHs) were used in the bead-based ALISSA for thedetection of BoNT/A. BoNT-specific VHH antibodies were provided by Dr.Charles Shoemaker of Tufts Cummings School of Veterinary Medicine (Maass2007; Tremblay 2010). In the ALISSA for BoNT/A-detection in spiked serumsamples, the VHHs JDY-46 and JEP-2 performed similarly as did themonoclonal antibody (mAb) 5A20.4 provided by Dr. James Marks (Universityof California at San Francisco). However, compared to the mAb, assaylinearity and sensitivity were inferior when using the VHHs (FIG. 52 A).

All VHHs tested were fusion proteins of E. coli thioredoxin (Trx) withthe antigen-binding domain (VHH) of a BoNT-specific alpaca single chainantibody. With the goal to improve the ALISSA limit of detection (LOD)and to design a chemical anchor that would tightly bind the affinityreagent to a bead or column surface while directing the antigen-bindingdomain away from the surface, the ALISSA performance of multivalent VHHmultimers with aminogroup-rich peptide appendices was determined. Forexample, the ALISSA performance of bivalent VHH molecules that containtwo BoNT-binding VHH domains was explored (H7 and C2, FIG. 52 B). Otherbi- and tri-valent VHH multimers consisted of Dr. Shoemaker's H7, D12(JIA-44) and H7, B5, C2 (JIA-31) molecules, respectively (FIG. 53 A)(Mukherjee 2012). ALISSA with these multivalent VHHs showed improvedlinearity and a LOD of ˜10 attomolar for JIA-44 and 1 femtomolar forJIA-31. H7 and D12 target different epitopes on the BoNT/A LC surface,while C2 has only weak LC-binding and associates strongly with theBoNT/A heavy chain (HC) (Gu 2012). This may explain the superior ALISSAresults obtained with the H7/D12 VHH heterodimer. Kinetic analysis ofJIA-44 revealed a binding constant of ˜65 pM for BoNT/A LC (FIG. 53 B,C). VHHs with decalysine peptide extensions to provide consistentamino-rich chemical anchor points are currently being designed. Ascyanogen bromide activation of sepharose is used to couple VHHs orantibodies to ALISSA beads, a similar aminogroup-reactive chemistry willbe used for the microcolumns which will be developed in conjunction withthe fully automated BoNT ALISSA detector.

Affinity Maturation of VHHs.

The VHHs provided by Dr. Shoemaker were derived from a cDNA library ofBoNT-immunized alpacas. The VHHs had their original natural bindingaffinity for BoNT/A, because none of the VHHs had been affinity maturedthus far. Therefore, a system for affinity maturation of BoNT/ALC-specific VHHs by modifying Wittrup's yeast surface display approachwas established (Boder 1997). In the original approach, a fusion proteinof Saccharomyces cerevisiae Aga2p, an HA tag, a scFv, and a c-myc tag isexpressed in yeast and displayed on its surface, whereby the Aga2pportion of the construct is bound via two disulfide bonds to cellwall-embedded Aga1p (Boder 1997). In the present method, the scFv-codingDNA sequence was replaced with that of the H7 VHH provided by Dr.Shoemaker. Additionally, a labeling technique with FITC-BoNT/A LC andAlexa Fluor^(647nm)-conjugated anti-c-Myc antibody was developed thatallows for detection and sorting of VHH-displaying yeast cells using aFluorescence Activated Cell Sorter (FACS) (FIG. 54 A). Error-prone PCRwas performed to introduce random mutations into the VHH, and thensingle cell selection was used to isolate cells that produced highlevels of VHH (c-Myc^(high)) at high affinity (FITC-LC^(high)) intosingle wells of medium containing 96-well plates (FIGS. 54 B and C). Atleast seven cycles of affinity selection for mutated VHHs will beperformed.

Screening for VHHs Directed to BoNT/B.

To extend the ALISSA detection to BoNT serotype B, eleven VHH proteinsfrom Dr. Shoemaker were screened to identify further suitable affinityreagents. The VHHs JGA-3, JFZ-28 appeared to yield the best performancecharacteristics in terms of dynamic range and LOD. Attomolar detectionlimits were achieved in bead based ALISSA using pooled human serum witha dilution series of BoNT/B LC (FIG. 55).

Microcolumn-Based ALISSA.

The ALISSA technology was further optimized for use with pipette tipcolumns (FIG. 56 A). The pipette tip columns were affinity microcolumnsthat contained a bead-based immunomatrix with immobilized BoNT antibodymounted into disposable pipette tips (FIG. 56 B). The antibodiesselected for the experiments were tested by Dr. Nedelkov at IntrinsicBioprobes Inc. for antigen binding after immobilization viaamine-reactive groups using a BiaCore chips and a surface plasmonresonance instrument. The chemistry used for immobilization on BiaCorechips was the same as for immobilization in the microfluidic affinitycolumns. Interestingly, the data suggested that some antibodies canreadily be utilized for the ALISSA after direct immobilization via aminegroups instead of using an immobilization matrix of protein NG-coatedbeads. An ALISSA utilizing affinity pipettes with microcolumns mountedinto disposable pipette tips was performed for detection of BoNT/A (FIG.56 A). Briefly, a biological sample was added to an affinity pipette tipwith a microcolumn containing the immobilized BoNT/A antibodyimmunomatrix. BoNT/A from the biological sample was bound and enrichedon the immunomatrix while the non-specific molecules (flow-through) werewashed away. Next, fluorogenic BoNT/A specific peptide was added to theaffinity pipette tip containing the bound BoNT/A. Upon substratecleavage and subsequent release of unquenched fluorophore, thefluorescence of the sample was read using a waveguide sensor. Theaffinity pipette tips can be used in conjunction with an electricmultichannel pipettor (FIG. 56 B, left panel) and also a fully-automatedrobotic pipetting workstation (FIG. 56 B, right panel).

The ALISSA with affinity microcolumns was tested using increasingamounts of BoNT/A light chain in pooled human serum (FIG. 57 A). Dextranglass columns from Intrinsic Bioprobes, Inc. (acquired by Thermo FischerScientific) were used for the microcolumns. The affinity microcolumnimmunomatrix contained the 5A20.4 (anti BoNT/A light chain) antibody.Results demonstrated that an increasing amount of BoNT/A-cleavablesubstrate, #201a, was cleaved upon the addition of increasingconcentrations of BoNT/A (FIG. 57A). Additionally, the ALISSA withaffinity microcolumns was tested using several different controls toverify the specific binding and activity of BoNT/A (FIGS. 57 B-E). Thecontrol experiments demonstrate that the cleavage of the BoNT/A specificsubstrate is due to BoNT/A activity as BoNT/A specifically binds theimmunomatrix conjugated with BoNT/A antibodies.

Additionally, a microcolumn robotic pipetting workstation (Versette,Thermo Fischer Scientific) was obtained for the development of anautomated microcolumn based BoNT ALISSA detection system. Affinity tipsthat hold microcolumns were designed that fit the Versette pipetteworkstation. The Versette workstation was used for fully automatedALISSAs (FIG. 56B, right panel). The system was programmed to conductmicrocolumn-enrichment of BoNT/A LC from spiked samples of human serum.Subsequently, the pipetting station performed automated washes of thecolumns, followed by enzymatic reaction with fluorogenic BoNT/Acleavable substrates. The approach is still under development, but anexample of a successful ALISSA run is shown in FIG. 57 F. Thisnon-optimized version of a microcolumn-based automated ALISSA canreadily detect 100 fmolar BoNT/A LC concentrations in 150 μL-sizedsamples of spiked human serum within 8 hours. In one embodiment theaffinity microcolumns may also be optimized to contain optimized VHHproteins generated from affinity maturation of BoNT/A and BoNT/Bspecific VHH reagents using the yeast display technique describedpreviously.

These results demonstrate a robust and fully automated ALISSA used forthe detection of BoNT. This method is expected to improve public healthand safety by offering a means to detect botulinum neurotoxins inbiological samples in a sensitive, fast, and inexpensive fashion.

The examples disclosed herein are provided to better illustrate theclaimed invention and are not to be interpreted as limiting the scope ofthe invention. To the extent that specific materials are mentioned, itis merely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention. All references cited herein are incorporated byreference as if fully set forth herein.

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What is claimed is:
 1. A method for detecting the presence of a BoNTtoxin in a sample comprising: a) exposing the sample putativelycontaining a BoNT toxin to an enrichment matrix comprising a substratefusion protein capable of eliciting a detectable luminogenic signalfollowing modification by the BoNT toxin, the exposure occurring underconditions permitting the modification of the substrate fusion proteinby the BoNT toxin, the substrate fusion protein comprising a BoNTcleavage site wherein: (i) if the BoNT toxin is BoNT/A, the substratefusion protein comprises a fragment of the synaptosome-associatedprotein of 25 kDa (SNAP25) containing the BoNT/A cleavage site; (ii) ifthe BoNT toxin is BoNT/B, the substrate fusion protein comprises afragment of vesicle-associated membrane protein (VAMP) containing theBoNT/B cleavage site; (iii) if the BoNT toxin is BoNT/C, the substratefusion protein comprises a fragment of VAMP containing the BoNT/Ccleavage site or a fragment of SNAP25 containing the BoNT/C cleavagesite; (iv) if the BoNT toxin is BoNT/D, the substrate fusion proteincomprises a fragment of VAMP containing the BoNT/D cleavage site; (v) ifthe BoNT toxin is BoNT/E, the substrate fusion protein comprises afragment of SNAP25 containing the BoNT/E cleavage site; (vi) if the BoNTtoxin is BoNT/F, the substrate fusion protein comprises a fragment ofVAMP containing the BoNT/F cleavage site; and (vii) if the BoNT toxin isBoNT/G, the substrate fusion protein comprises a fragment of VAMPcontaining the BoNT/G cleavage site; and b) detecting the presence ofthe BoNT toxin by measuring a change in light emission in the sample. 2.The method of claim 1 wherein the substrate fusion protein comprises:one or more firefly luciferase proteins or a fragment thereof, and aBoNT cleavable SNAP25 sequence, a BoNT cleavable VAMP sequence, or acombination thereof.
 3. The method of claim 2 wherein the substratefusion protein comprises amino acid residues 187-206 of SNAP25 and aminoacid residues 1-550 of SEQ ID NO:
 26. 4. The method of claim 2 whereinthe substrate fusion protein further comprises one or more peptidesselected from the group consisting of a positive control cleavage site,an affinity tag, a linker, and a lysine rich anchor.
 5. The method ofclaim 4 wherein the positive control cleavage site comprises SEQ ID NO:33, the affinity tag comprises SEQ ID NO: 32, the linker comprises SEQID NO: 34 or SEQ ID NO: 35, and the lysine rich anchor comprises SEQ IDNO: 30 or SEQ ID NO:
 31. 6. The method of claim 4 wherein the substratefusion protein is SEQ ID NO:
 25. 7. The method of claim 2 wherein thesubstrate fusion protein comprises overlapping luciferase fragmentsinterrupted by a BoNT cleavage sequence.
 8. The method of claim 7wherein the substrate fusion protein comprises a first fireflyluciferase fragment of amino acid residues 1-475 or amino acid residues1-478 of SEQ ID NO: 26; a BoNT cleavable SNAP25 sequence; and a secondfirefly luciferase fragment of amino acid residues 265-550 or amino acidresidues 476-550 of SEQ ID NO:
 26. 9. The method of claim 7 wherein thesubstrate fusion protein comprises a first firefly luciferase fragmentof amino acid residues 1-475 or amino acid residues 1-478 of SEQ ID NO:26; a first binding domain; a BoNT cleavable SNAP25 sequence; a secondfirefly luciferase fragment of amino acid residues 265-550 or amino acidresidues 476-550 of SEQ ID NO: 26; and a second binding domain capableof binding to the first binding domain.
 10. The method of claim 9wherein the first binding domain is selected from the group consistingof glutathione, glutathione S-transferase, and IgG FC-region; and thesecond binding domain is selected from the group consisting ofglutathione S-transferase, and protein A.
 11. A method for detecting thepresence of a BoNT toxin in a sample comprising: a) exposing the sampleputatively containing a BoNT toxin to an enrichment matrix comprising asubstrate fusion protein comprising one or more firefly luciferaseproteins or a fragment thereof, the substrate fusion protein capable ofbeing modified by the BoNT toxin, the exposure occurring underconditions permitting the modification of the substrate fusion proteinby the BoNT toxin, the substrate fusion protein comprising a BoNTcleavage site wherein: (i) if the BoNT toxin is BoNT/A, the substratefusion protein comprises a fragment of the synaptosome-associatedprotein of 25 kDa (SNAP25) containing the BoNT/A cleavage site; (ii) ifthe BoNT toxin is BoNT/B, the substrate fusion protein comprises afragment of vesicle-associated membrane protein (VAMP) containing theBoNT/B cleavage site; (iii) if the BoNT toxin is BoNT/C, the substratefusion protein comprises a fragment of VAMP containing the BoNT/Ccleavage site or a fragment of SNAP25 containing the BoNT/C cleavagesite; (iv) if the BoNT toxin is BoNT/D, the substrate fusion proteincomprises a fragment of VAMP containing the BoNT/D cleavage site; (v) ifthe BoNT toxin is BoNT/E, the substrate fusion protein comprises afragment of SNAP25 containing the BoNT/E cleavage site; (vi) if the BoNTtoxin is BoNT/F, the substrate fusion protein comprises a fragment ofVAMP containing the BoNT/F cleavage site; and (vii) if the BoNT toxin isBoNT/G, the substrate fusion protein comprises a fragment of VAMPcontaining the BoNT/G cleavage site; and b) exposing the modifiedsubstrate fusion protein to a detection fusion protein, the exposureoccurring under conditions permitting the binding of the substratefusion protein to the detection fusion protein; and c) detecting thepresence of the BoNT toxin by measuring a change in light emission. 12.The method of claim 11 wherein the substrate fusion protein comprises afirst binding domain; a first firefly luciferase fragment; a BoNTcleavable SNAP25 sequence or a BoNT cleavable VAMP sequence; and thedetection fusion protein comprises a second firefly luciferase fragment;a second binding domain capable of binding to the first binding domainof the substrate fusion protein.
 13. The method of claim 12 wherein thefirst firefly luciferase fragment comprises amino acid residues 265-550or amino acid residues 476 to 550 of SEQ ID NO: 26 and the secondfirefly luciferase fragment comprises amino acid residues 1-475 or aminoacid residues 1-478 of SEQ ID NO:
 26. 14. The method of claim 12 whereinthe substrate fusion protein and the detection fusion protein areimmobilized onto beads.
 15. A method for detecting the presence of aBoNT toxin in a sample comprising: a) exposing the sample putativelycontaining a BoNT toxin to an enrichment matrix provided in a columncomprising a substrate fusion protein comprising one or more fireflyluciferase proteins or a fragment thereof, the substrate fusion proteincapable of eliciting a detectable luminogenic signal followingmodification by the BoNT toxin, the exposure occurring under conditionspermitting the modification of the substrate fusion protein by the BoNTtoxin, the substrate fusion protein further comprising a BoNT cleavagesite wherein: (i) if the BoNT toxin is BoNT/A, the substrate fusionprotein comprises a fragment of the synaptosome-associated protein of 25kDa (SNAP25) containing the BoNT/A cleavage site; (ii) if the BoNT toxinis BoNT/B, the substrate fusion protein comprises a fragment ofvesicle-associated membrane protein (VAMP) containing the BoNT/Bcleavage site; (iii) if the BoNT toxin is BoNT/C, the substrate fusionprotein comprises a fragment of VAMP containing the BoNT/C cleavage siteor a fragment of SNAP25 containing the BoNT/C cleavage site; (iv) if theBoNT toxin is BoNT/D, the substrate fusion protein comprises a fragmentof VAMP containing the BoNT/D cleavage site; (v) if the BoNT toxin isBoNT/E, the substrate fusion protein comprises a fragment of SNAP25containing the BoNT/E cleavage site; (vi) if the BoNT toxin is BoNT/F,the substrate fusion protein comprises a fragment of VAMP containing theBoNT/F cleavage site; and (vii) if the BoNT toxin is BoNT/G, thesubstrate fusion protein comprises a fragment of VAMP containing theBoNT/G cleavage site; and b) detecting the presence of the BoNT toxin bymeasuring a change in light emission of the sample.
 16. The method ofclaim 15 wherein the substrate fusion protein is selected from the groupconsisting of: (i) amino acid residues 187-206 of SNAP25 and a fireflyluciferase fragment of amino acid residues 1-550 of SEQ ID NO: 26; (ii)a first firefly luciferase fragment of amino acid residues 1-475 oramino acid residues 1-478 of SEQ ID NO: 26, a BoNT cleavable SNAP25sequence, and a second firefly luciferase fragment of amino acidresidues 265-550 or amino acid residues 476 to 550 of SEQ ID NO: 26; and(iii) SEQ ID NO:
 25. 17. The method of claim 15 wherein the substratefusion protein further comprises one or more peptides selected from thegroup consisting of a positive control cleavage site, an affinity tag, alinker, and a lysine rich anchor.
 18. The method of claim 15 wherein thesubstrate fusion protein comprises: a first firefly luciferase fragment;a first binding domain; a BoNT cleavable SNAP25 sequence or a BoNTcleavable VAMP sequence; and a second firefly luciferase fragment; and asecond binding domain capable of binding to the first binding domain ofthe substrate fusion protein.
 19. The method of claim 18 wherein thefirst binding domain is selected from the group consisting ofglutathione, glutathione S-transferase, and IgG FC-region; and thesecond binding domain is selected from the group consisting ofglutathione S-transferase and protein A.
 20. The method of claim 15wherein the column is selected from the group comprising: affinitymicrocolumns, and pipette tip columns containing mounted affinitymicrocolumns; and the columns are used in conjunction with a highthroughput system.